Method of producing a Mad polypeptide

ABSTRACT

Nucleic acid molecules capable of hybridizing under stringent conditions to the nucleotide sequence residing between positions 1 and 453 of the max cDNAs shown in FIG. 2, or to the nucleotide sequence residing between positions 148 and 810 of the mad cDNAs shown in FIG. 14. The Max polypeptide when associated with the Myc or Mad polypeptide is capable of binding to nucleotide sequences containing CACGTG.

This invention was made with government support under grants T32CA09437,RO1 CA20525, PO1 CA28151 and CA57138 awarded by the National Institutesof Health. The government has certain rights in the invention.

This application is a continuation-in-part of application Ser. No.07/756,195, filed Sep. 9, 1991, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to genetic engineering involvingrecombinant DNA technology, and particularly to the identification of abHLP-Zip polypeptide, termed Max, that specifically associates withc-Myc polypeptides such that the Myc-Max complex binds to DNA in asequence specific manner, and a polypeptide termed Mad, thatspecifically associated with Max and is a competitive inhibitor of Mycbinding to Max.

BACKGROUND OF THE INVENTION

The products of the MYC family of protooncogenes, including c-Myc,N-Myc, and L-Myc proteins, function in cell proliferation,differentiation, and neoplastic disease (1; see the appended Citations).However, there is as yet no consensus as to the molecular mechanism bywhich Myc mediates its biological effects. The Myc proteins are nuclearphosphoproteins with short half-lives and nonspecific DNA-bindingactivities (2). Functionally important regions exist as both the aminoand carboxyl termini of the c-Myc protein (3-5). Indeed, thecarboxyl-terminal 85 amino acids of the Myc family proteins sharesignificant sequence similarity with two classes of transcriptionfactors, the basic region helix-loop-helix (bHLH) and basic regionleucine zipper (bZip) proteins, both of which have basic regionsadjacent to their dimerization domains. The bHLH family includes over 60proteins in vertebrates, yeast, plants, and insects; many, if not all,exhibit nuclear localization, are sequence-specific DNA-bindingproteins, and function as transcriptional regulators(6). The region ofsequence similarity shared to Myc and other proteins in this class is acritical determinant of function and contains a stretch of basic aminoacids followed by two putative amphipathic a helices that flank anW-type loop (7,8). Studies of several other bHLH proteins havedemonstrated that the HLH region mediates formation of homo- orheterodimers, which in turn permits the basic regions to form a DNAcontact surface (9-11). Myc family proteins differ from the bHLH familyin that adjacent and carboxyl-terminal to their bHLH motif is another ahelix that contains a heptad repeat of leucine residues. This structureis characteristic of the dimerization domains of the bZip family oftranscriptional regulators (12). The array of nonpolar amino acids formsa hydrophobic face long the amphipathic helic, facilitating specificassociation of bZip proteins through a parallel coiled-coil interaction(13). Dimerization is critical for DNA binding (14, 15).

For c-Myc there is substantial evidence that the bHLH region and theadjacent leucine zipper motif are functionally important. Deletionswithin these regions result in loss of alteration of transformingactivity (3, 16) as well as reduction of the capacity of autoregulateendogenous myc expression and to inhibit cell differentiation (4, 5). Inaddition, a bacterially expressed fusion protein that contains thebHLH-Zip domains of c-Myc has sequence-specific DNA-binding activity(17).

It is also of interest to consider the myc oncogene in the context oftumor suppressor genes since, at least on theoretical grounds, it isprecisely the proliferation-inducing effects of myc that one wouldexpect to be opposed by genes of the tumor suppressor class. The notionthat myc oncogene function is linked to cell proliferation is nowsupported by multiple lines of evidence. Much of this evidence has beensummarized in recent reviews (18,19) and will be briefly reiteratedhere. First, c-myc expression is strongly correlated with cell growth.During exponential growth of many different cell types, c-myc-encodedmRNA and protein synthesis is maintained at a constant level throughoutthe cell cycle (20,21). By contrast, c-myc expression is essentiallyundetectable in quiescent (G₀) cells and in most, but not all,terminally differentiated cell types. The down-regulation of c-mycexpression during differentiation is likely to be a critical event sinceforced expression of exogenous c-myc blocks the induced differentiationof erythroleukemia cells and adipocytes (22, 23) while anti-senseinhibition of c-myc expression in HL60 cells leads directly todifferentiation (24).

On the other hand, the entry into the cell cycle of quiescent cells isinvariably accompanied by a large transient burst of c-myc expressionwithin hours of mitogenic stimulation of both hematopoietic andnonhematopoietic cell types (25). Indeed, c-myc is prototypical of theclass of immediate early response genes encoding labile mRNAs which canbe induced (or superinduced) in the presence of protein synthesisinhibitors. That myc expression is important for entry into the cellcycle is suggested by experiments utilizing c-myc anti-senseoligonucletides, which appear to block the entry of mitogenicallystimulated human T cells into S phase but not into the G₁ phase of thecell cycle (26). Recent experiments using an artificially "activatable"c-myc-encoded protein (c-Myc) have demonstrated that quiescentfibroblasts can be made to enter the cell cycle following activation ofc-Myc. Amazingly, this occurs in the absence of the induction of theother major early response genes, including jun and fos (27). Thus,c-myc expression may be sufficient for entry of G₀ cells into the cellcycle.

Further support for the idea that Myc function is strongly linked tocell proliferation and differentiation comes from the vast amount ofdata demonstrating an association between the deregulation of myc familygene expression and neoplasia (for reviews, see 28-30). Oncogenicactivation of myc by retroviral capture, promoter/enhancer insertion,gene amplification, and chromosomal translocations all appear to lead toabnormal and uncontrolled proliferation of numerous cell types. Whilethese events frequency result in myc overexpression, they also result ina loss of the normal regulatory elements that control normal mycexpression. A great deal of work has demonstrated that myc expression isnormally regulated at multiple levels (for recent review, see 31), andit is the loss of such regulation which is believed to result inuncontrolled cell proliferation and a reduced capacity for terminaldifferentiation.

Although it is indisputable that Myc is involved in cell proliferation,it is less clear whether the functions of tumor suppressor genes, whichare often through to act as negative growth regulators (see 32 forreview), actually impinge directly on Myc function. One potentialexample of interaction between myc function and tumor suppressor geneactivity has come from studies demonstrating that treatment of anepithelial cell line with TGF-β results in transcriptional repression ofc-myc which is reversible by agents (adenovirus E1A, SV40 R antigen)that sequester the Rb gene product (33,34). While these data do notnecessarily indicate a direct interaction between Myc and Rb they atleast hint at the possibility that the functional pathways of these twogene products may be intertwined. In addition, it is possible that Mycmight interact directly with an as yet uncharacterized tumor suppressorprotein. It is clear that more details concerning the molecularmechanism of Myc function are required in order to explore more fullythe possibility of direct interaction between Myc and tumor suppressorgene products. One approach is to define the interactions of Myc proteinwith other cellular proteins, as well as with nucleic acids. Suchstudies may help to elucidate Myc's molecular function and reveal thecircuitry through which proliferation suppression factors may interactwith Myc.

The biological importance of and structural similarities in the carboxylterminus of c-Myc suggest that Myc functions as a component of anoligomeric complex. While Myc self-association has been demonstratedwith relatively high concentrations of bacterially expressed Myc protein(35), coprecipitation, chemical crosslinking, and dimerization motifchimeras fail to demonstrate homodimerization of Myc under physiologicalconditions (1, 36, 37). Because functionally relevant interactions occuramong members of the bHLH and bZip classes (9, 15, 38, 39), and c-Mychas not yet been found to associate with members of either group (10,15, 16), we hypothesized that Myc function may depend on heterotypicinteraction with an unknown protein. We now describe the cloning of sucha Myc binding factor, termed Max, and its regulatory factor, termed Mad.

SUMMARY OF THE INVENTION

The invention provides isolated nucleic acid molecules capable ofhybridizing under stringent conditions to the nucleotide sequenceresiding between positions 1 and 453 of the max cDNA shown in FIGS.2A-B, and the nucleotide sequence residing between positions 1 and 1002of the mad cDNA shown in FIG. 14. In the preferred embodiment, suchisolated nucleic acid molecules encode Max polypeptides thatspecifically associate with Myc polypeptides, and Mad polypeptides thatassociate with Max, respectively. Such a Max polypeptide, either alone(homodimerized) or when associated with the Myc polypeptide, is capableof binding to a nucleotide sequence containing CACGTG as an activationcomplex. Max associated with Mad is also capable of binding CACGTG, butas a repressor complex. In a related embodiment, such isolated nucleicacid molecules encode polypeptides that are recognized by antibodiesthat bind to the Max polypeptide shown in FIGS. 2C-D, and the Madpolypeptide shown in FIGS. 14A-B.

The subject nucleic acid molecule can be operably linked to suitablecontrol sequences in recombinant expression vectors. The recombinantexpression vectors are used to transfect or transduce cells, such thatthe engineered cells produce a Max polypeptide that specificallyassociates with a Myc polypeptide, or a Mad polypeptide that associateswith Max. Polypeptides so produced are generally characterized asencoded by a gene sequence capable of hybridizing under stringentconditions to the nucleotide sequence residing between positions 1 and43 of the max cDNA shown in FIGS. 2A-B, or residing between positions148 and 810 of the mad cDNA shown in FIGS. 14A-B.

The invention also provides isolated polypeptide Max:Myc complexes, inwhich a Max polypeptide is associated with a Myc polypeptides, andMad:Max complexes, in which Max is associated with Mad. The Mycpolypeptide may be encoded by the c-myc, L-myc, N-myc, or v-mycprotooncogenes. Such isolated polypeptide Max:Myc or Mad:Max complexesare capable of binding to the nucleotide sequence CACGTG.

The invention also provides isolated DNA molecules capable ofhybridizing under stringent conditions to: the nucleotide sequenceresiding between positions 88 and 123 of the helix 1 region of the maxcDNA shown in FIGS. 2A-B; the nucleotide sequence residing betweenpositions 142 and 186 of the helix 2 region of the max cDNA shown inFIGS. 2A-B; the basic region sequence residing between positions 43 and81 of the max cDNA shown in FIGS. 2A-B; and, the leucine zipper regionresiding between position 210 and 270 shown in FIGS. 2A-B. Also providedare isolated DNA molecules encoding polypeptides that can specificallyassociated with Myc polypeptides but that do not bind to the nucleotidesequence CACGTG; such mutant DNA molecules are capable of hybridizing tothe nucleotide sequence residing between positions 82 and 453 shown inFIGS. 2A-B, but not to the basic region residing between positions 43and 81 shown in FIGS. 2A-B.

The invention also provides isolated DNA molecules capable ofhybridizing under stringent conditions to: the nucleotide sequenceresiding between positions 355 and 399 of the helix I region of the madcDNA shown in FIGS. 14A-B; the nucleotide sequence residing betweenpositions 418 and 471 of the helix II region of the mad cDNA shown inFIGS. 14A-B, the basic region sequence residing between positions 319and 354 of the mad cDNA shown in FIGS. 14A-B; and, the heptadhydrophobic zipper region sequences residing between positions 472 and597 of the mad cDNA shown in FIGS. 14A-B. Also provided are isolated DNAmolecules encoding mutant Mad polypeptides, that can specificallyassociate with Max polypeptides at a higher affinity that a non-mutantMad, and mutant Mad polypeptides can competitively inhibit Myc bindingto Max more at a lower concentration than non-mutant Mad polypeptides.Other isolated DNA molecules are provided that encode mutant Madpolypeptides that have binding affinity for Max, but when in complexwith Max may fail to bind to the nucleotide sequence CACGTG, i.e.,mutant in the DNA binding site domain contributed to the complex by Mad.In one example, the mutant Mad polypeptides are encoded by DNA moleculesthat are hybridizing to the nucleotide sequence residing betweenpositions 148 and 810 shown in FIGS. 14A-B but not to the basic regionresiding between positions 319 and 354 shown in FIGS. 14A-B.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A diagrammatically represents the Myc fusion protein used toscreen for Max, as described in Example 1;

FIGS. 1B-C, show representative specific binding of the Myc fusionprotein to Max polypeptide expressed by plaques, as described in Example1;

FIGS. 2A-D present the nucleotide and amino acid sequences of Max andthe organization of Max cDNA, as described in Example 2;

FIGS. 3A-B diagrammatically delineate and compare the amino acidsequences of the HLH region and basic zipper (bZip) regions of the Mycand Max poly peptides, as discussed in Example 2;

FIGS. 3C schematically depicts the alignment of the HLH regions in Mycand Max involved in protein-protein interactions that form the Myc:Maxcomplex, and the alignment of Myc and Max bHLH-Zip regions involved inbinding of Myc and Max and the Myc:Max complex to promoter sequences inDNA, as describe in Example 2;

FIG. 4 shows representative binding of Myc with Max to form the Myc:Maxcomplex isolated by affinity chromatography, as described in Example 3;

FIG. 4B illustrates structural requirements in Myc for formation of theMyc:Max complex utilizing altered forms of Myc, as described in Example3; and

FIG. 4C presents the results of control experiments showing thespecificity of the Myc:Max interaction, as described in Example 3;

FIG. 5A shows binding of Max to Myc family members, as described inExample 3;

FIG. 5B presents the results of experiments showing the specificity ofthe Myc:Max interaction and failure of Myc to interact with other HLHbZip polypeptides, as described in Example 3;

FIG. 6 shows formation of the Myc:Max complex by Myc and Maxpolypeptides synthesized in vitro and isolation of the complex byimmunoprecipitation and SDS-PAGE, as described in Example 4;

FIG. 7A shows the binding of the isolated purified Myc:Max complex tothe core consensus nucleotide sequence CACGTG, as described in Example4;

FIG. 7B illustrates the specificity of the binding of isolated purifiedMyc:Max complex to the core consensus nucleotide sequence, as describedin Example 4;

FIG. 7C delineates the protein structural requirements for binding ofthe Myc:Max complex to the core consensus nucleotide sequence, asdescribed in Example 4;

FIG. 8A shows detection and isolation of Max p21 and Max p22polypeptides from mammalian cells, as described in Example 5;

FIGS. 8B-E show the two-dimensional peptide maps of radiolabeled Max p21and Max p22 polypeptides, as described in Example 5;

FIG. 8F compares Max p21 and Max p22 polypeptides purified frommammalian cells with synthetic Max polypeptides resulting from in vitrotranslation, as described in Example 5;

FIGS. 9A-H show detection and localization of Max in mammalian cells byimmunofluororescence assays, as described in Example 5;

FIG. 9I portrays phosphorylation of Max by protein kinases involved inregulation of cell growth and replication, as described in Example 5;

FIG. 10 presents the results of studies showing the stability of Max p21and Max p22 in cells, as described in Example 5;

FIGS. 11A-C depict the relationship between the expression of the MaxmRNA and polypeptides and growth and replication of mammalian cells, asdescribed in Example 5;

FIG. 12A depicts representative isolation and purification of Myc:Maxcomplexes from mammalian cells and compares the results ofhigh-stringency (HS) and low-stringency (LS) isolation conditions, asdescribed in Example 5; and,

FIGS. 12B-D depict representative isolation and purification of Myc:Maxcomplexes from mammalian cells at low-stringency and purification of theMyc polypeptides from the isolated complexes at high stringency fordeterming Myc stability with a Myc:Max complex, as described in Example5.

FIG. 13A-C show autoradiograms of gels in which altered electrophoreticmobility of Max:oligonucleotide complexes was used to identifypolypeptides that bound specifically with Max. The ability of Maxpurified from sf9 cells to bind the CM-1 binding site was assayed by anelectrophoretic mobility shift assay as described below, (FIGS. 13A and13B).

FIG. 13A shows an autoradiograms of an a gel in which decreasedelectrophoretic mobility of Max:Max:oligonucleotide complexes wasobserved in the presence of antibodies to max(αmax). Max binding to DNAwas assayed in the absence ("-") or the presence of a Max-specificanti-peptide antiserum. "αMax+block" indicates the inclusion of bothαMax and the immunizing peptide in the Max/oligonucleotide bindingreaction.

FIG. 13B shows an autoradiogram of a gel in which the electrophoreticmobility of Max:Max homodimeric complexes with CM-1 oligonucleotide (oran unrelated MREA oligonucleotide) was assayed. Levels of Max:Maxcomplexes were assayed in the presence of unlabeled CM-1 or MREA. Thelevels of Max:Max were unchanged as the amount of MREA was increasedfrom 25 ng to 100 ng in the assay, but when CM-1 was increased from 25ng to 100 ng the levels of Max:Max complexes decreased. (The amount ofcompeting oligonucleotide is given in ng in the box at the top of eachlane of the gel. "-" denotes no unlabeled oligonucleotide in the bindingreaction.) The position of the free probe and the Max homodimer mobilityshift is as marked. The Max:Max* asterisk denotes the electrophoreticmobility of the antibody:Max:Max complex.

FIG. 13C shows an autoradiogram of ³² P-labeled Max binding to a filtercontaining phage lysates from different gt11 lambda clones. Clone Max 14was identified in the FIREST SERIES OF EXAMPLES as being a bindingpartner for Myc. λ1 encoded a lacZ fusion protein with no specific Maxbinding activity and served as a negative control in this experiment.λ10, 11, and 26 encoded lacZ fused to potential Max binding partners.

FIGS. 14A-B shows the nucleotide (Seq. ID No.: 5) sequence of humanMad-1 cDNA and the amino acid sequence (Seq. ID No.: 6) encoded thereby.(The nucleotide and the amino acid sequence of the coding ration of the3.2 kb human Mad-1 cDNA from the WI26 gt10 library is shown.) Nucleotidepositions are indicated. Amino acid positions are denoted by bold facednumbers and in frame stop codons are shown. The basic region homology isboxed and the positions of the positively charged residues in thisregion are marked by "+". The shaded boxes locate helix I and helix II.The amino acids that form the hydrophobic heptad repeat (i.e., positions108 to 150) are given in bold underlined text. The region rich in acidicamino acids is located between amino acids 152 and 189.

FIGS. 15A-B show a comparison of the amino acid sequence of Mad-1 withother b-HLH proteins.

FIGS. 15A-B show the predicted amino acid sequence of Mad-1 as itcompares with other members of the b-HLH family of transcription factorsand to the b-HLH consensus sequence ("Cons."). The amino acids aredenoted by the single letter code. The Drosophila proteins EMC(extramacrocheatae) and hairy were found to be most similar to Mad-1 insearches of the data base while TFE3 and USF both recognize the same DNAbinding site (CACGTG) as Myc and Max. The matches to the b-HLH consensusare shaded and the residues that form a heptad repeat of hydrophobicamino acids are shaded and boxed. The shaded and cross-hatched bar atthe bottom of FIG. 15A depicts a generalized organizational structurefor a b-HLH-zipper protein.

FIG. 15C shows the organizational structure of the Mad-1, Max and Mycpolypeptides. The numbers indicate amino acid position. The basicregion, helix-loop-helix, and leucine zipper homologies are as indicatedby cross-hatching, shading, and dashed-lines, respectively.

FIGS. 16A-F, show autoradiograms of gels in which Mad-1 was mixed withother b-HLH polypeptides to test its binding specificity as described inthe THIRD SERIES OF EXAMPLES, below. The results of the electrophoreticgel shift experiments show the specificity of Mad-1 binding to Max. Inthis experiment, RNAs encoding the proteins given at the top of eachpanel (e.g., Max RNA and Max 9 RNA in FIG. 16 Panel A) were translatedand labeled with ³⁵ S-methionine in vitro in the presence of eitherpurified glutathione-S-transferase (GST) or glutathione-S-transferasefused in frame to baboon Mad-1 cDNA (Mad). The proteins bound by GST orGST-Mad were analyzed on SDS polyacrylamide gels. The lanes marked "-"indicated translation products obtained in the absence of added purifiedGST or GST-Mad protein. In FIG. 16B the arrows mark the position ofeither the ABR Max or Max9 or ALZ Max polypeptide. The position ofmolecular weight markers (in kD) are given at the right of each panel.

FIGS. 17A-C shows autoradiograms of gels from experiments designed todetermine the DNA binding specificity of the Mad:Max heterodimer. Theability of Mad-1 to bind DNA and interact with Max and Myc was examinedby the electrophoretic mobility shift assay. Purified fusion proteins,GST-Mad (FIG. 17A), and GST-C92Myc (FIG. 17B) were tested alone or inthe presence of Max for binding to the CM-1 oligonucleotide. Non-fusionGST protein was used as a control. The protein(s) present in the bindingreaction is indicated at the top of each gel lane in FIGS. 17A-B. As acontrol, the specificity of the electrophoretic mobility shift wasassayed by including antibodies to either Max (αMax), GST (αGST) or Myc(αMyc) were added to the binding reaction. The activity of theseantibodies was inhibited by adding the appropriate immunoglobulin to thebinding reaction ("+block"). The lanes marked "-" had no additionalprotein present in the binding reaction. The position of eachprotein-DNA complex and the unbound probe is given. The "*" asterisk,(e.g., GST-Mad:Max*), indicates electrophoretic mobility of the controlantibody complex. A diagram of the organizational structures of the GSTpolypeptides used in this experiment is shown below FIG. 17C.

FIGS. 18A-B show an autoradiogram of a gel in which formation and DNAbinding of Max:Max or Mad:Max complexes (FIG. 18A); and, the Myc:Maxheterodimer (FIG. 18B) were evaluated as a function of increasingconcentrations of Max (right directed arrows; "increasing Max" at thetop of the gel lanes); at a constant concentration of either GST-Mad(i.e., 30 ng) or GST-C92Myc (i.e., 25 ng). The formation of Mad:Maxheterodimeric complexes was found to be favored over the Max:Maxhomodimer. (Increasing amounts of Max were assayed for DNA binding tothe CM-1 oligonucleotide by the electrophoretic mobility shift assayeither alone or in the presence of 30 ng GST-Mad or 25 ng GST-C92Myc).When assayed alone Max in the binding reactions was increased in roughly2 fold increments from 0.3 ng to 10 ng. The same amounts of Max weretested with the indicated amount of fusion protein. In the lane marked"-" there was no protein in the binding reaction. The positions of theunbound probe and the protein:DNA complexes are indicated.

FIG. 19 shows an autoradiogram of a gel in which the binding ofphosphorylated Mad was tested for formation of complexes with Max andbinding to DNA. The results show that CKII phosphorylation does notaffect the DNA binding of the Mad:Max heterodimer. (Max or Max treatedwith CKII was tested for DNA binding to the CM-1 oligonucleotide by theelectrophoretic mobility shift assay presence of GST or GST-Mad. Theproteins in the binding reactions are given at the top of the FIGURE. Inthe lanes marked "CKII-ATP" or "CKII+ATP Max" was treated with CKIIeither in the absence or the presence of ATP, respectively, prior toinclusion in the DNA binding reaction. The positions of the free probeand the protein DNA complexes are indicated.)

FIGS. 20A-C show autoradiograms of gels in which the binding affinity ofMyc:Max and Mad:Max to DNA was compared in electrophoretic mobilityshift assays. The proteins present in the binding reactions is given atthe top of each gel lane. ("-") indicates the absence of protein in thebinding reaction. The positions of the protein:DNA complex and theunbound probe is given at the right of each panel.

FIG. 20A shows an autoradiogram of Max:Max, Myc:Max and Mad:Max bindingto DNA. The results show similar binding affinities of Myc:Max andMad:Max for the CM-1 DNA probe. (The DNA binding characteristics of thepurified histidine tagged Mad was assayed FIG. 20A).

FIG. 20B shows an autoradiogram of Myc:Max binding to DNA as a functionof increasing concentrations of Mad in the binding reaction. (At aconstant amount of GST-C92Myc:Max (6 ng:2 ng) increasing amounts of Madwere added to the incubation mixture, (right arrow), in 2 foldincrements starting at 1.8 ng and ending at 30 ng.)

FIG. 20C shows an autoradiogram of Mad:Max binding to DNA as a functionof increasing concentrations of Myc in the binding reaction. (At aconstant amount of Mad:Max (7.5 ng:2 ng) increasing amounts ofGST-C92Myc were added to the incubation mixture, (right arrow) in 2 foldincrements starting from 1.6 ng and ending at 50 ng.)

FIG. 21 shows a schematic diagram depicting the binding interactions ofthe Myc, Max and Mad-1 polypeptides, and the DNA binding sites in therespective complexes to the specific CACGTG nucleotide sequence motif inDNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The Myc protooncogene family has been implicated in cell proliferation,differentiation, and neoplasia, but its mechanism of function at themolecular level has been unknown. The carboxyl terminus of Myc familyproteins contains a basic region helix-loop-helix leucine zipper motif(bHLH-Zip), which has DNA-binding activity and has been predicted tomediate protein-protein interactions. As described in the First Seriesof Examples below, the bHLH-Zip region of c-Myc was used to screen acomplementary DNA (cDNA) expression library, and a bHLH-Zip protein,termed Max, was identified. Max specifically associated with c-Myc,N-Myc, and L-Myc proteins, but not with a number of other bHLH, bZip, orbHLH-Zip proteins. The interaction between Max and c-Myc was dependenton the integrity of the c-Myc HLH-Zip domain, but not on the basicregion or other sequences outside the domain. Furthermore, the Myc-Maxcomplex bound to DNA in a sequence-specific manner under conditionswhere neither Max nor Myc exhibited appreciable binding. The DNA-bindingactivity of the complex was dependent on both the dimerization domainand the basic region of c-Myc. These results suggest that Myc familyproteins undergo a restricted set of interactions in the cell and maybelong to the more general class of eukaryotic DNA-binding transcriptionfactors.

Mad associates specifically with Max, but not with Myc or other b-HLHtranscription regulatory factors. Mad may be a competitor proteinassociating with Max and forming a negative regulatory complex termedMad:Max. The levels of Mad, Max, and/or Myc may determine activation orrepression of genes regulated by transcription regulatory factorsbinding at the CACGTG motif in DNA.

As described in the Second Series of Examples below, Myc and Max areassociated in vivo, and essentially all of the newly synthesized Myc canbe detected in a complex with Max. The stability of Myc protein isunchanged by its association with Max. In vivo, Max is shown to be ahighly stable nuclear phosphoprotein whose levels of expression areequivalent in quiescent, mitogen-stimulated, and cycling cells. The rateof Myc biosynthesis is therefore likely to be a limiting step in theformation of Myc:Max complexes.

As described in the Third Series of Examples below, addition of Mad topreformed Max:Myc complexes causes dissociation of the complex withformation of Mad:Max complexes, similarly, addition of Myc to Mad:Maxcomplexes causes dissociation and formation of Max:Myc.

The invention provides, in a representative embodiment, an isolatednucleic acid molecule (DNA or RNA) that is capable of hybridizing understringent conditions to the nucleotide sequence residing betweenpositions 1 and 453 of the max cDNA shown in FIGS. 2A-B. By "capable ofhybridizing under stringent conditions" is meant annealing to a cDNAshown in FIGS. 2A-B (i.e., with or without the 27-mer insertion shownbetween base positions 36 and 37), or its complementary strand, understandard conditions, e.g., high temperature and/or low salt content,which tend to disfavor hybridization. A suitable protocol (involving0.1×SSC, 68° C. for 2 hours) is described in Maniatis, T., et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,1982, at pages 387-389. The nucleic acid so annealed may be one of thetwo max cDNAs shown in FIGS. 2A-B, portions thereof, or of any otheralternatively spliced forms or max-related cDNAs and genes.

The subject nucleic acid molecule preferably encodes a Max polypeptidethat can associate with a Myc polypeptide. The Max polypeptide whenassociated with the Myc polypeptide is capable of binding to thenucleotide sequence CACGTG.

The subject Max polypeptides are produced by operably linking theisolated nucleic acid molecule to suitable control sequences inrecombinant expression vectors. Cells transfected or transduced withsuch recombinant expression vectors are capable of expressing theencoded polypeptides. Such polypeptides are generally encoded by a genesequence capable of hybridizing under stringent conditions to thenucleotide sequence(s) residing between positions 1 and 453 of the maxcDNA shown in FIGS. 2A-B. Such polypeptides preferably also associatewith Myc polypeptides.

In a related embodiment, the invention provides an isolated polypeptideMax:Myc complex in which a Max polypeptide is associated with a Mycpolypeptide. The Myc polypeptide may be encoded by the c-myc, L-myc,N-myc, or v-myc protooncogenes. The isolated polypeptide Max:Myc complexis generally capable of binding to the nucleotide sequence CACGTG.

In related embodiments, the invention provides isolated DNA moleculescapable of hybridizing under stringent conditions to the nucleotidesequence residing between positions 88 and 123 of the helix 1 region ofthe max cDNA shown in FIGS. 2A-B; the nucleotide sequence residingbetween positions 142 and 186 of the helix 2 region of the max cDNAshown in FIGS. 2A-B; the basic region sequence residing betweenpositions 43 and 81 of the max cDNA shown in FIGS. 2A-B; and, theleucine zipper region residing between position 210 and 270 shown inFIGS. 2A-B. Mutant DNA molecules are also provided. In a representativeexample, the mutant DNA molecule encodes a polypeptide that canspecifically associate with a Myc polypeptide but that does not bind tothe nucleotide sequence CACGTG; this particular mutant DNA molecule iscapable of hybridizing to the nucleotide sequence residing betweenpositions 82 and 453 shown in FIGS. 2A-B but not to the basic regionresiding between positions 43 and 81 shown in FIGS. 2A-B.

In addition to its evident value as a research reagent, the followingpotential uses relating to Max are contemplated:

1. Changes in the levels of Max and especially of the Myc:Max complex asa diagnostic or prognostic tool for diverse types of cancer. This mightinvolve standard protocols using a reagent, such as a monclonalantibody, which recognizes the Myc:Max complex but not Myc or Max alone(or homodimerized). The max gene resides on chromosome 14 q22-24(unpublished), and changes in this region might be implicated inneoplasia.

2. Interference with formation or maintenance of the Myc:Max complex asa means of retarding neoplasia. This might occur through specificantibodies (following cellular uptake) or with chemical reagents (suchas specific peptides or drugs) which interfere with interaction betweenthe helix-loop-helix-zipper domains of the two proteins. Design of suchreagents may entail knowledge of the three-dimensional structure of bothMyc and Max and the complex(es). Studies using NMR and X-raycrystallography are contemplated.

3. A "dominant negative Max" which is capable of forming a complex withMyc but is nonfunctional might be used to influence neoplasia. One suchMax has a deletion or substitution of the Max basic region involved inDNA binding.

4. If Max itself is a negative regulator of cell growth then increasingthe levels of Max through different vectors might phenotypically opposean increase in Myc levels. Likewise, decreasing Max levels, e.g.,through anti-sense vectors, might also influence cell growth.

5. Max appears to be expressed throughout embryonic development (M. W.King, unpublished), and therefore alterations in Max levels mightinfluence key events in embryogenesis.

6. The Max:Myc, Max:Max, and possibly Myc:Myc complexes are likely tobind DNA sequences that are involved in regulation of transcriptionand/or DNA replication. In either case Myc and Max may regulate genesthat themselves are involved in DNA replication and cell proliferation.Any of these genes and their products could lead to new insights intohow to regulate growth and may be subject to analysis and intervention.

The invention also provides, in a representative embodiment, an isolatednucleic acid molecule (DNA or RNA) that is capable of hybridizing understringent conditions to the nucleotide sequence residing betweenpositions 1 and 1002 of the mad cDNA shown in FIGS. 14A-B. By "capableof hybridizing under stringent conditions" is meant annealing to a cDNAshown in FIGS. 14A-B, or its complementary strand, under standardconditions, e.g., high temperature and/or low salt content, which tendto disfavor hybridization. A suitable protocol (involving 0.1×SSC, 68°C. for 2 hours) is described in Maniatis, T., et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, at pages387-389. The nucleic acid so annealed may be one of the two mad cDNAsshown in FIG. 14, portions thereof, or of any other alternativelyspliced forms of mad-related cDNAs or genes.

The subject nucleic acid molecule preferably encodes a Mad polypeptidethat can associate with a Max polypeptide. Mad is an inhibitor of Myc(and other bHLH proteins binding to Max) at least two levels. First, theMad polypeptide is capable of competitively inhibiting binding of Myc toMax. Second, both Mad:Max and Myc:Max bind to the same CACGTG nucleotidesequence, such that Mad:Max complex is a competitive inhibitor ofMyc:Max. It is reasoned likely by the inventors that once bound to DNAthe Mad:Max complex acts a negative transcription regulator, whileMyc:Max is a positive activator.

The subject Mad polypeptides are produced by operably linking theisolated nucleic acid molecule to suitable control sequences inrecombinant expression vectors. Cells transfected or transduced withsuch recombinant expression vectors are capable of expressing theencoded polypeptides. Such polypeptides are generally encoded by a genesequence capable of hybridizing under stringent conditions to thenucleotide sequence(s) residing between positions 148 and 809 of the madgene shown in FIGS. 14A-B. Such polypeptides also preferably associatewith Max polypeptides.

In a related embodiment, the invention provides an isolated polypeptideMad:Max complex in which a Mad polypeptide is associated with a Maxpolypeptide. The Max polypeptide may be encoded by a nucleotide sequencecapable of hybridizing with the nucleotide sequence of FIGS. 2A-B, andthe Mad polypeptide may be encoded by a nucleotide sequence capable ofhybridizing with the nucleotide sequence of FIGS. 14A-B. The isolatedpolypeptide Mad:Max complex is capable of binding to the nucleotidesequence CACGTG.

In related embodiments, the invention provides isolated DNA moleculescapable of hybridizing under stringent conditions to: the nucleotidesequence residing between positions 355 and 399 of the helix I region ofthe mad cDNA shown in FIG. 14; the nucleotide sequence residing betweenpositions 418 and 471 of the helix II region of the mad cDNA shown inFIGS. 14A-B; the basic region sequence residing between positions 319and 354 of the mad cDNA shown in FIGS. 14A-B; and, the heptadhydrophobic zipper region sequences residing between positions 472 and597 of the mad cDNA shown in FIGS. 14A-B. Mutant DNA molecules are alsoprovided. In a representative example, the mutant DNA molecule encodes aMad polypeptide that can specifically associate with a Max polypeptideand prevent Max binding to Myc; this particular mutant DNA molecule iscapable of hybridizing to the nucleotide sequence residing betweenpositions 148 and 810 shown in FIGS. 14A-B but not to the basic regionresiding between positions 319 and 354 shown in FIGS. 14A-B. A secondmutant DNA molecule encodes a Mad polypeptide that can specificallyassociate with a Max polypeptide and inhibit binding of the mutantMad:Max complex to DNA at the transcription regulatory CACGTG nucleotidesequence; this particular mutant DNA molecule is capable of hybridizingto the nucleotide sequence residing between positions 319 and 471 shownin FIGS. 14A-B but not to the region residing between positions 472 and597 shown in FIGS. 14A-B.

In addition to its evident value as a research reagent, the followingpotential uses relating to Mad are contemplated:

1. Changes in the levels of Mad and especially of the Mad:Max complexcould serves as a diagnostic or prognostic tool for diverse types ofcancer. This might involve standard protocols using a reagent, such as amonclonal antibody, that recognizes the Mad:Max complex but not Mad,Max, Max:Myc, or Max:Max (i.e., homodimerized). Rearrangements of themad gene chromosomal region may be implicated in neoplasia, sinceinactivation of the repressor activity of Mad may result in decreasedregulatory control over endogenous positive regulatory elements, i.e.,positive regulators that bind Max. In this respect, Mad may be similarto members of the tumor suppressor gene families, e.g., Rb and p53.

2. Interference with formation or maintenance of the Myc:Max complexcould serve as a means of retarding neoplasia. Mad competes binding ofthe b-HLH zipper domains of Myc proteins to Max; and Mad:Max complexesmay act as a repressor of Max:Myc activation of transcription orcellular replication. Increasing the levels of Mad polypeptides in acell (e.g., over-expressing Mad) may counteract the activating effectsof Myc. Alternatively, mutant Mad reagents (and mimetic compounds)having higher binding affinity for Max may interfere with Max binding toMyc.

3. A "dominant repressor Mad" which is capable of forming a complex withMax that either inhibits formation of Max:Myc complexes, or inhibitsMax:Myc binding to CACGTG regions in DNA, may thereby influenceneoplasia. One such Mad has substitutions in one or more nucleotides inthe basic HLH region or zipper region of Mad that is involved in Maxbinding. Such HLH-region substitution or zipper-region substitution(s)in Mad preferably increase the binding affinity of Mad for Max, or ofthe Mad:Max complex for CACGTG regions in DNA.

4. If Mad itself is a negative regulator of Max-mediated cell growth (orMax:Myc-mediated cell growth) then increasing the levels of Mad in acell by using gene transfer vectors may phenotypically oppose thetransformed phenotype of a cell. In a related aspect, Mad (or Mad:Maxcomplexes) may drive terminal differentiation in a cell, and genetransfer designed to increase the levels of Mad in a cell may be usefulfor driving terminal differentiation of a transformed (e.g., cancer)cell. Alternatively, decreasing Mad levels in a terminallydifferentiated cell, (e.g., through antisense vectors) could be usefulto promote cell growth in vitro and tissue regeneration in vivo. Forexample, smooth muscle cells having a terminally differentiatedphenotype may be induced to grow in vitro for prolonged periods of timeusing antisense Mad vectors to increase the level of expression of Madin these cells.

5. Mad may be expressed as a competitive inhibitor of growth promotingelements in cells that specifically associate with Max, e.g., Myc, andMad:Max complexes may act as negative repressor of such Max-bindinggrowth promoting elements in a terminally differentiated cell. In thisregard, alterations in Mad levels could influence certain key events incellular differentiation.

6. The Mad:Max, Max:Myc, Max:Max, and possibly Myc:Myc complexes arelikely to bind DNA sequences that are involved in regulation oftranscription and/or DNA replication. In any case Myc, Max, and Mad mayregulate genes that themselves are involved in DNA replication and cellproliferation. The genes regulated by Max:Mad, Max:Max, and Max:Myc, andthe polypeptide products of such regulated genes, may lead to newinsights into regulation of cell growth and terminal differentiation ofcells. As such, the latter genes regulated by Max complexes may beimportant targets for drug development because selected chemical,polypeptide, and antisense inhibitors of expression of Mad and Max mayalter cell growth and phenotype.

FIRST SERIES OF EXAMPLES

Max is a helix-loop-helix-zipper protein that associates in vitro withMyc family proteins to form a sequence-specific DNA binding complex.

EXAMPLE 1 Functional Cloning of a Myc Binding Protein

Biologically interactive proteins have been identified by functionalcloning (40). This work encouraged us to use the c-Myc b-HLH-Zip regionto identify proteins from a λgt11 cDNA expression library that interactwith Myc. We prepared a construct that consisted of thecarboxyl-terminal 92-amino acid residues of human c-Myc fused to thecarboxyl terminus of glutathione-S-transferase (GST-MycC92). FIG. 1A isa diagram of the GST-MycC92 fusion protein used for iodination andscreening, wherein the following abbreviations apply: CKII, caseinkinase II phosphorylation site; BR, basic region; HLH, helix-loop-helix;and LZ, leucine zipper. This bacterially expressed fusion protein wassoluble, easily purified, and contained 17 tryrosines as potentialiodination sites (only one of which lies within the Myc segment).Furthermore, this protein, which was used to identify a specificDNA-binding sequence for c-Myc (17), contains the complete b-HLH-Zipregion and thus should have the minimal structure required for DNAbinding and protein interaction.

GST-MycC92 was expressed in Escherichia coli, purified byglutathione-agarose affinity chromatography and ¹²⁵ I-labeled to highspecific activity. Specifically, GST-MycC92 fusion protein was expressedfrom a pGEX-2T plasmid (Pharmacia) that contained the 570-base pair AvaII-Eco RI fragment of human c-myc cDNA clone (0/1) ligated into the SMAI-Eco RI cloning sites. Fusion protein was purified as described [D. B.Smith and K. S. Johnson, Gene 67, 31 (1988)]. GST (50 mg) or GST-MycC92(50 mg) were ¹²⁵ I-labeled to high specific activity (72 mCi/mg) withIodobeads (Pierce) as recommended by the manufacturer [M. A. K.Markwell, Anal. Biochem. 125, 427 (1982)].

For cloning we used a random-primed λgt11 expression library derivedfrom a baboon lymphoblastoid cell line. The λgt11 expression library wasconstructed from the baboon lymphoid cell line 594S as described [R. L.Idzerda et al., Proc. Natl. Acad. Sci. U.S.A. 86, 4659 (1989)]. Phagefrom this library produce nearly full-length β-galactosidase proteinsfused with the open reading frames of the directionally cloned c-DNAs.More than 10⁶ plaques were screened for their ability to interact with¹²⁵ I-labeled GST-MycC92. Specifically, the 594S λgt11 library wasplated on the Y1088 bacterial strain. As plaques became visible,β-galactosidase fusion protein expression was induced by overlaying thelawns with IPTG [isopropyl β-D-thiogalactopyranoside (10mM)]-impregnated nitrocellulose filters (Amersham; Hybond C Extra).Transfer of released proteins was allowed to proceed overnight. Filterswere marked, rinsed to remove bacterial debris, and blocked with 5percent dry milk in HND buffer [20 mM Hepes, pH 7.2, 50 mM NaCl, 0.1percent NP- 40, and 5 mM dithiothreitol (DTT)] for 1 hour at 4° C. ¹²⁵I-labeled GST or GST-MycC92 (100 ng/ml, about 3 nM) was added to thefilters in HND buffer supplemented with 1 percent dry milk. After a4-hour incubation on a rotating platform at 4° C., filters were rapidlywashed seven times with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄.7H₂ O, 1.4 mM KH₂ PO₄, pH 7.3) that contained 0.2 percent TritonX-100 (room temperature). Filters wrapped in plastic were exposed toX-ray film for 3 hours to overnight [see (40) for related protocols].

Several potential positive plaques were identified, two of which (Max11and Max14) survived multiple rounds of plaque purification.Representative results are shown in FIGS. 1B-C, wherein: At top left,secondary plating of five putative positives demonstrates the reactivityof two of the primary plaques, Max11 and Max14. At top right, as anegative control, GST was labeled to a similar specific activity andcompared with GST-MycC92 for binding to Max14 plaques.

Because the observed binding might have been mediated by the GSTsequences in the fusion protein, the plaques were probed with GST ¹²⁵I-labeled to the same specific activity as GST-MycC92. Only the GSTfusion protein that contained c-Myc, and not GST alone, reacted with theMax14 plaques. Representative results are shown in FIG. 1B, wherein: Atbottom, binding of GST-MycC92 to Max14 plaques was assayed with orwithout affinity purified carboxyl terminal-specific anti-Myc (Ab) orpeptide immunogen (peptide). In addition, affinity-purified antibodiesto the 12 carboxyl-terminal amino acids of human c-Myc (anti-Myc) (41)partially blocked the binding of GST-MycC92 to the plaques in a mannerthat was prevented by addition of the peptide immunogen (FIG. 1B).

To confirm that the association of GST-MycC92 with Max11 and Max14 wasattributable to specific protein-protein interaction, Max11 and Max14lysogen proteins were fractionated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), transferred to nitrocellulose filters, andsubjected to protein blotting with ¹²⁵ I-labeled GST-MycC92. WhileGST-MycC92 failed to bind to β-galactosidase alone, it did bind toβ-galactosidase fusion proteins in both Max11 and Max14 lysates (16).These results indicate that the Myc-containing segment of GST-MycC92specifically interacts with the protein products encoded by Max11 andMax14 cDNAs.

EXAMPLE 2 Identification of a Helix-Loop-Helix-Zipper Domain in Max

Nucleotide sequence analysis of the inserts from both of the GST-MycC92reactive λgt11 phages demonstrated that Max11 and Max14 encode the sameprotein as defined by the β-galactosidase open reading frame.Specifically, sequence analysis of Max11 and Max14 clones, along withMax clones derived from a Manca λgt10 library, was performed by thedideoxy method [F. Sanger, S. Miklen, A. F. Coulson, Proc. Natl. Acad.Sci. U.S.A. 74, 5463 (1977)]. The 513-nucleotide sequence presented(FIGS. 2A-B) was constructed from two overlapping Manca cDNA clones.

FIGS. 2A-B show nucleotide and amino acid sequences of Max. The Max openreading frame, as generated from overlapping Manca cell cDNAs (human),encodes a 151-amino acid polypeptide. The 9-amino acid insertion foundin several PCR clones is shown above the inverted triangle. Helix I andhelix II of the b-HLH homology region are underlined, while thehydrophobic heptad repeat, which extends from helix II into the zipperregion, are in bold face and underscored. Basic and acidic regions areidentified by their charge (+ or -), and termination codons are markedby asterisks.

Both Max11 and Max14 appear to be partial, overlapping cDNAs. Max11 andMax14 encode 124 and 131 amino acids, respectively, between the junctionwith β-galactosidase and a TAA termination codon. In retrospect, it isnot surprising to have cloned only two functional inserts from the 10⁶plaques screened. Size selection of the cDNA inserts along with thepresence of an in-frame stop codon located two codons 5' to theinitiating AUG (see FIGS. 2A-B) limits the number of potentiallyfunctional points of lacZ fusion (that is, those that contain an intactHLH-Zip region) to 40. For comparison, screening of the 594S λgt11library with a c-Myc carboxyl-terminal specific antiserum identifiedonly 12 immunoreactive plaques.

Subsequent isolation of several overlapping cDNAs from a Manca (humanBurkitt's lymphoma cell line) λgt10 library permitted deduction of anapparently complete open reading frame for Max that encodes 151 residues(FIGS. 2A-B). This is based on the assignment of an AUG in relativelygood context for translational initiation (42) that was preceded by anupstream, in-frame termination codon. This amino acid sequence probablyrepresents the complete Max open reading frame, because antibodies toMax (anti-Max) were used to immunoprecipitate a cellular protein thatcomigrated with, and produced an identical tryptic peptide map as the invitro translation product of the Max cDNA (16). Sequencing ofMax-specific polymerase chain reaction (PCR) products from Manca cDNAsrevealed a putative variant form of Max that differed only by a 9-aminoacid insertion amino-terminal to the basic region (FIGS. 2C-D). In theexperiments described below, we utilized a Max cDNA that lacked thisinsertion.

FIGS. 3A-C show the structure of the Max protein and its sequencesimilarity shared with other bHLH-Zip proteins. FIGS. 3A-B show regionsof sequence similarity shared with other bHLH transcription factors. TheMax b-HLH-Zip region is compared and contrasted to that of other b-HLHproteins found in humans (MyoD, E12, AP-4, USF, c-Myc, L-Myc, andN-Myc), insects (As-C), plants (Lc), and yeast (CBF-1). Shaded regionsidentify residues that fit the consensus as derived from the known b-HLHfamily (43) (F=L, I, V, M; W=F, L, I, Y). Boxes denote the heptad repeatof hydrophobic residues, which extends from helix II into the putativeleucine zipper.

Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp;E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn;P, Pro, Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

A computer search of the protein database IPIR Version 25) revealedsequence similarity between a segment of the Max open reading frame andthe b-HLH proteins, including members of the Myc family (FIGS. 3A-B).The Max sequence in this region represents a nearly exact match with theHLH consensus (FIGS. 3A-B bottom) (43). The similarity among Max and theb-HLH proteins also extends in the amino-terminal direction into a basicregion of Max. The Max sequence just carboxyl terminal to helix IIcontains a series of hydrophobic amino acid residues, three of which areleucines, spaced seven residues apart (FIGS. 3A-B boxed residues).Helical wheel analysis (12) of this region suggests that the amphipathichelix II may extend into and beyond the three leucines. These leucinesand the other nonpolar residues might form a hydrophobic face similar tothat in the leucine zipper proteins.

We have also shown that a 15-amino-acid deletion of the basic regionabolishes the capacity of c-myc to cotransform Rat-1 cells icollaboration with the brc-abl oncogene. In contrast, a 9-amino-aciddetection of a neighboring region, the CKII phosphorylation site (seeFIG. 3C), has little effect on cotransformation (data not shown). Theseexperiments lend further support to the notion that the b-HLH-Zip regionof Myc is critical for function.

FIG. 3C presents a schematic representation of Myc and Max proteins asaligned by their regions of sequence similarity (stippled boxes).Abbreviations are used to designate the casein kinase II phosphorylationsite (CKII), basic region (BR), helix-loop-helix (HLH), leucine zipper(LZ), and acidic region (AR). Numbering corresponds to their respectiveamino acid sequences.

The Max polypeptide sequence is hydrophilic in nature. More thanone-third of its residues are charged, and the most abundant amino acidis serine (14 percent) Max contains no cysteines. Despite a predictedmolecular size of 17,200 daltons, Max, like Myc, exhibits aberrantelectrophoretic mobility in SDS-polyacrylamide gels (see FIG. 5A). Theorganization of the Max cDNA protein coding sequence and the relativeextents of the basic, HLH, zipper, and carboxyl-terminal regions aredepicted in FIG. 3C. The similarity of Max with other bHLH proteins islimited to the Max bHLH region, and the sequences of baboon and humanMax do not correspond to those of any previously identified protein.

EXAMPLE 3 Specific interaction of Max with Myc family proteins

The present of putative bHLH and leucine zipper domains in Max suggeststhat Max interacted with a similar region of GST-MycC92 in thefunctional library screening. To further investigate the potential ofthis region of Max to associate with Myc, we developed an affinitychromatography assay in which the Max protein, linked to a solidsupport, was used to test for binding of full-length, wild-type Myc anda series of mutant Myc proteins.

A set of deletion and point mutations were introduced into a wild-typehuman c-myc cDNA (pHLmyc 0/1) that contained the complete c-Myc openreading frame (44). Specifically, oligonucleotide-directed mutagenesiswas used to generate a variety of mutant Myc proteins: ΔC89, deletion ofthe carboxyl-terminal 89 resides (deleted amino acids 351 to 439, addedArg-Arg-Thr-Ser); ΔBR, deletion of the basic region (deleted amino acids353 to 367); ΔCKII, deletion of the casin kinase II phosphorylation sitelocated 5' of the basic region (deleted amino acids 346 to 354); ΔHelix1, deletion of Δhelix 1 (deletion of amino acids 368 to 381); ΔLZ,deletion of the leucine zipper (deleted amino acids 416 to 439); BR21M,replacement the basic region of Myc with that of MyoD (replaced Mycamino acids 347 to 367 with MyoD amino acids 102 to 122); BR21MDLZ,double mutant that consists of BR21M and D LZ; and ProZip, replacementof Leu (amino acid residue 420) with Pro (from A. J. Street). Deletionof sequences 5' to the Pvu II site in the 0/1 cDNA resulted in D N100;translation from this construct initiates at the first internalmethionine (amino acid 101). Numbering corresponds to the amino acidsequence of human c-Myc [R. Watt et al., Nature 303, 725 (1983)].Dideoxy sequencing and immunoprecipitation of in vitro translationproducts were used to confirm the identity of each construct.

RNAs prepared from the normal and mutated clones by in vitrotranscription were translated in a rabbit reticulocyte lysate togenerate c-Myc proteins labeled with [³⁵ S]methionine. Specifically, invitro transcription and translation were performed under conditionsrecommended by the Promega Protocols and Applications Guide. [³⁵S]Methionine labeled proteins were produced from each of the followingvectors: pVZ1-Max11/13/14, pBluescribe vectors that contained the mutantMyc constructs, pU313S (L-Myc) (from K. Alitalo), pNmycB (N-Myc) (fromR. Bernards), pV2C11a (MyoD) (from A. Lassar) (11), E12R (E12) (from C.Murre) (7), pBS-B065 (myogenin) (from W. Wright) (45), tal-SP6pGem (Tal)(from R. Baer) (46), pJun7'8 (Jun) (from R. Turner), pSP65fos1B (Fos)(from T. Curran), D12.2 (USF) (from R. Roeder) (47), and T7bAP-4 (AP-4)(from Y-F. Hu and R. Tijan) (48). Programmed reticulocyte lysate (1 ml)was subjected directly to SDS-PAGE, or lysates (20 ml) were diluted intoHND buffer (400 ml) that contained bovine serum albumin (BSA) (10mg/ml). Half of this dilution was incubated with either GST orGST-Max124 beads [approximately 5 mg of fusion protein adsorbed to 10 mlof glutathione-Sepharose (Pharmacial)] for 1 hour at 4° C. The resin wasthen washed four times with PBS that contained NP-40 (0.1 percent) atroom temperature. The bound proteins were eluted with SDS-containingsample buffer and subjected to SDS-PAGE and autoradiography.

A fusion protein that contained the carboxyl-terminal 124 amino acids ofMax (GST-MaxC124) was coupled to glutathione-Sepharose beads.GST-MaxC124 was constructed by insertion of the Ava II-Eco RI fragmentof Max14 into the Sma I site of pGEX-3X expression vector (Pharmacia).The resulting fusion protein had the 124 carboxyl-terminal amino acidsof Max in frame with GST sequences. Fusion protein was purified asdescribed in Example 1, supra. The labeled in vitro translation products(FIG. 4A, top panel) were incubated with GST-MaxC124 or GST resin,washed under low stringency conditions, and the bound material waseluted with SDS and analyzed by SDS-PAGE as described above.

FIG. 4 presents structural requirements for Myc-Max association.Wild-type (0/1) or mutant forms of the c-Myc protein were assayed fortheir ability to associate with the HLH-Zip motif of Max. After in vitrotranslation, programmed reticulocyte lysate (RL) was subjected directlyto SDS-PAGE analysis (1 ml), or the sample (10 ml) was purified on GSTor GST-MaxC124 affinity columns and the bound material was subjected toSDS-PAGE as described above. Mutations: ΔN100, deletion of theamino-terminal 100 amino acids of c-Myc (this mutation removes the twoalternative initiation codons that normally are translated to producethe p64-p67 doublet); ΔC89, deletion of the carboxyl-terminal 89 aminoacids; ΔLZ, deletion of the leucine zipper, ΔBR, basic region deletion,ΔHelix 1, helix I deletion, ΔCKII, casein kinase II phosphorylation sitedeletion (49); ProZip, proline was substituted for leucine at position 2of the zipper region; BR21M, the basic region of C-Myc was replaced withthat of MyoD). Migration of the molecular size markers are indicated.

None of the c-myc translation products bound to GST alone (FIG. 4C,bottom panel), while GST MaxC124 resin retained the wild-type c-Mycproteins, p64 and p67 (0/1; FIG. 4, middle panel). The ability of c-Mycprotein to interact with Max was dependent on an intact carboxylterminus, as deletion of the carboxyl-terminal 89-amino acid residues(ΔC89) completely abolished binding to Max, while deletion of 100residues at the amino terminus (ΔN100) had no effect (FIG. 4B, middlepanel). To ascertain what regions within the carboxyl-terminal domainwere required for the binding, we examined a series of mutations.Neither deletion of the Myc basic region (ΔBR), its substitution withthe MyoD basic region (BR21M), or deletion of one of the CKIIphosphorylation sites (CKII, just amino terminal to the basic region)(49) had any effect on association with Max. In contrast, binding to Maxwas inhibited by deletion of either c-Myc helix I or the leucine zipper,as well as by substitution of a helix-disrupting proline residue for thesecond leucine in the zipper. These results suggest that full-lengthc-Myc interacts with the carboxyl-terminal region of Max and that thisassociation is mediated by the c-Myc HLH-Zip domain.

Because N-Myc and L-Myc also have b-HLH-Zip regions at their carboxyltermini (7, 12), we assessed their ability to bind Max. Results areshown in FIGS. 5A-B which presents an analysis of Max binding to Mycfamily members (5A), and to b-HLH, bZip, and b-HLH-Zip (5B) proteins. Invitro transcription and translation were used to produce proteinslabeled with [³⁵ S]methionine. After in vitro translation, programmedreticulocyte lysate (RL) was subjected directly to SDS-PAGE analysis (1ml), or the lysate (10 ml) was purified on GST or GST-Max C124 (Max)affinity columns and the bound material was subjected to SDS-PAGE asdescribed above. Molecular size markers migrated as indicated on theSDS-PAGE analysis.

[³⁵ S]Methionine-labeled in vitro translation products, generated fromN-myc cDNAs, bound to GST-MaxC124 resin with the same efficienty as thec-Myc protein (FIG. 5A). In vitro translated, full-length Max proteinalso bound to the Max-containing resin suggesting that Max mayhomo-oligomerize. Neither the Myc family proteins nor Max bound GSTalone. To test the possibility that any protein that contains an HLH orleucine zipper motif might associate with Max, we obtained cDNAs thatencode other transcription factors and determined the ability of theirin vitro translation products to bind to GST-MaxC124 resin. Categoriesof transcription factors examined included MyoD, E12, Ta1, and myogenin,all of which possess b-HLH domains (8, 45, 46); Fos and Jun, each ofwhich contain a leucine zipper (12); and AP-4 (48) and USF (47), whichcontain adjacent HLH and leucine zipper regions. Although none of theseproteins bound either GST or GST-MaxC124, specific interaction betweenc-Myc and GST MaxC124 was again observed (FIG. 5B). This assay is arather stringent test of association, because relatively low amounts oflabeled protein compete for binding with a large excess of maxhomodimers (or homo-oligomers). Furthermore, the reticulocyte lysate maycontaining competitors or inhibitors of binding. Therefore, theinability of specific proteins to interact with Max in the assay may notbe a reflection of the in vivo situation.

EXAMPLE 4 Formation of a Myc-Max complex with sequence-specificDNA-binding activity

Experiments in which bacterially expressed GST-MycC92 was used to selectpreferred DNA sequences from a pool of partially randomizedoligonucleotides have shown that c-Myc has specific DNA-binding activityfor the sequence CACGTG(17). Compared to other bHLH proteins used inthis assay, in vitro translated c-Myc bound relatively poorly to anoligonucleotide that contained this sequence (CM-1). These results mightbe explained in terms of inefficient homodimerization, inefficientbinding of homodimers to the CM-1 sequence, or both. Because Max iscapable of specifically associating with Myc, we tested the possibilitythat the Myc-Max heterocomplex might exhibit increased binding to CM-1compared to Myc alone.

For the DNA-binding assays, it was important to use full-length Myc andMax proteins in a soluble complex. Therefore, we first determinedwhether the full-length forms of both Max and c-Myc specificallyassociate in solution. The p64 and p67 c-Myc proteins and the p21 Maxprotein produced by in vitro translation of their respective cDNAs wererecognized by their cognate antisera (FIG. 6). Specifically, in vitrotranscripts from the c-Myc and Max vectors were added to a Promegareticulocyte lysate translation mixture and incubated for 1 hour at 30°C. c-Myc and Max (2:1) were mixed after translation, thus compensatingfor differences in the translational efficiencies of the two RNAspecies; association was allowed to proceed for 30 minutes at 30° C.,after which the lysates were diluted into PBS with 1 percent NP-40.Proteins were immunoprecipitated under these mild conditions withanti-Myc (5 mg of affinity purified) (41) or anti-Max (5 ml ofpolyclonal antisera to the GST-Max124 fusion protein (described above).Immunoprecipitations were blocked by the addition of the cognateimmunogen. Antigen-antibody complexes were isolated on proteinA-Sepharose beads (Sigma), and the pellets were washed five times withPBS that contained 1 percent NP-40. The [³⁵ S]Methionine labeled sampleswere analyzed by SDS-PAGE under reducing conditions.

FIG. 6 shows post-translational association of full-length Myc and Max.After separate in vitro translations, c-Myc and Max lysates were mixed,incubated for 30 minutes at 30° C., and immunoprecipitated with theindicated antibodies under the low stringency conditions describedabove. Anti-Myc was specific for the carboxyl-terminal 12 amino acids ofhuman c-Myc (41); anti-Max was raised against the GST-MaxC124 fusionprotein. Immunoprecipitated [³⁵ S]methionine labeled proteins wereresolved by SDS-PAGE.

Under low stringency immunoprecipitation conditions, anti-Myc failed torecognize Max and anti-Max failed to recognize c-Myc. However, when Maxand c-Myc were combined after translation, each antiserum precipitatedMax was well as c-Myc. The ability of a specific antiserum toprecipitate the two proteins after mixing is best explained by formationof Myc-Max complexes that are stable under the immunoprecipitationconditions. This idea is supported by the results of blockingexperiments, which demonstrate that coprecipitation of both proteinsoccurs only through the antigenic determinants of one of them (16). TheMyc mutants that fail to bind to truncated Max in the affinitychromatography experiments (FIG. 4) also did not associate withfull-length in vitro translated Max in the coimmunoprecipitation assay(16).

Having established that full-length Max and c-Myc associate in solution,we next determined whether the Myc-Max complex could bind a specific DNAsequence in a gel retardation assay. Specifically, Max or c-Myctranscripts were translated in vitro with nonradioactive methionine.Post-translational mixes were performed as in described above, and theresulting lysates were analyzed for binding to the synthetic CM-1oligonucleotide by the electrophoretic mobility shift assay [A. Revzin,BioTechniques 7, 346 (1989)]. Final conditions within a 25-ml bindingreaction were: 20 mM Hepes, pH 7.2, 50 mM KCl, 3 mM MgCl₂, 1 mM DTT, 1mM EDTA, 8 percent glycerol, 25 ng of sheared salmon sperm DNA as anonspecific competitor, 10 ml of programmed reticulocyte lysate, and 0.2ng of ³² P-labeled CM-1 oligo (17). The DNA-binding reaction was allowedto proceed at room temperature for 10 minutes. For antibody experiments,affinity purified anti-Myc or anti-Max (1 mg) was added for 10 minutesafter the formation of the nucleoprotein complex; the cognate immunogen(10 mg) blocked this supplemental shift. As competitors, double-strandedoligonucleotides were added at 1, 10, and 100 ng per reaction; the coresequence of the B1/B2 and CM-1 templates are 5'-CCCCCAACACCTGCTGCCTGA-3'and 5'-CCCCCACCACGTGGTGCCTGA-3', respectively (17). Protein-DNAcomplexes were resolved on a 5 percent acrylamide gel (50 mM tris base,50 mM borate, 1 mM EDTA), and gels were dried prior to autoradiography.

Incubation of an unprogrammed reticulocyte translation lysate with the³² P-labeled CM-1 oligonucleotide resulted in retardation of the probe(FIG. 7A). This binding appeared to be due to endogenous USF protein,which also recognizes the CM-1 sequence. Specifically, the backgroundbands in the gel retardation assays were due to endogenous USF bindingfactor activity, and binding of USF could be inhibited by the additionof the CM-1 probe. USF specifically binds to the CM-1 consensus [R. W.Carthew, L. A. Chodosh, P. A. Sharp, Cell 43, 439 (1985); M. Sawadogoand R. Roeder, ibid., p. 165; A. C. Lennard and J. M. Egly, EMBO J. 6,3027 (1987)] and is present in the reticulocyte lysates, as evidenced bythe ability of antibodies to USF to alter the mobility of these bands(L. Kretzner, unpublished data). Antibodies to USF were provided by M.Sawadoto.

When the translation lysates were programmed with Max RNA, no additionalbinding to the probe was detected, while lysates that contained c-Mycreproducibly showed a faint band of retarded probe (FIG. 7A).Retardation of the CM-1 probe was observed when reticulocyte lysate thatcontained both c-Myc and Max were used in the assay. That both c-Myc andMax proteins were bound to the retarded DNA probe was demonstrated bythe ability of both anti-Myc and anti-Max to decrease theelectrophoretic mobility of the bound probe. The specificity of thisantibody effect on mobility of the probe was confirmed by the fact thatit could be reversed for each antibody by addition of the cognateimmunogen (FIG. 7A). The specificity of binding to CM-1 was verified incompetition experiments in which a 5 fold excess of unlabeled CM-1 wassufficient to compete for binding by the Myc-Max complex. By contrast, a500-fold excess of an oligonucleotide (B1/B2) that contained a bindingsite for MyoD and E12 (50) and differed by only three nucleotides fromCM-1 was required to achieve a similar degree of competition (FIG. 7B).

Considered in additional detail, FIG. 7 presents analysis of Myc-Maxcomplex DNA-binding activity. FIG. 7A: the ability of in vitrotranslated Myc and Max proteins to bind to the CM-1 oligonucleotide(CACGTG core consensus) was assessed by electrophoretic mobility shiftassay. Post-translational mixes of Myc and Max were performed as inreference to FIG. 6. Lysates were incubated with ³² P-labeled CM-1 priorto resolution in a 5 percent acrylamide gel. In experiments whereantibodies were added, affinity purified antibody (1 mg) was added afterformation of the nucleoprotein complex to minimize steric interference.To block the antibody effect, cognate immunogen (1 mg) was added. Thepositions of probe specifically bound and further retarded by antibodyare indicated with asterisks. The arrow indicates free oligonucleotide.CM-1 oligonucleotide alone (probe) and unprogrammed reticulocyte lysate(RL) served as background controls. FIG. 7B: The specificity of theMyc-Max shift was tested by competition with 5-, 50-, and 500-foldexcess of unlabeled oligonucleotide. B1/B2 contains the 3' MCK enhancerbinding site for MyoD (CACGTG core consensus) and differs from CM-1 atonly three positions. FIG. 7C: Requirements for the formation of anucleoprotein complex. Various c-Myc mutants (see FIG. 4 discussion forabbreviations) were assayed for their ability to bind CM-1 inassociation with Max.

Binding of Myc to immobilized Max was dependent on the integrity of theHLH and leucine zipper domains (FIG. 4). To ascertain whether theassociation of Myc and Max in a nucleoprotein complex required the samesequences, some of the c-Myc mutants were examined for their ability tobind to CM-1 in a complex with Max. Specific binding to the CM-1 probeby Max and c-Myc was abolished when c-Myc mutants that lacked theputative leucine zipper domain or basic region were used in place ofwild-type c-Myc. By contrast, a c-Myc deletion mutant that did notdirectly affect the b-HLH-Zip region (such as DN100, which lacks 100amino-terminal residues of c-Myc), both associated with Max (FIGS. 5A-B)and bound to the CM-1 oligonucleotide. Therefore, loss of DNA bindingcorrelates with the inability of c-Myc mutants to associate with Max inthe binding assay. An exception to this is the basic region deletionmutant, which associated with Max but did not bind the CM-1 probe (FIGS.7A-C), a result that suggests a requirement for the basic region ofc-Myc in specific DNA binding but not in protein-protein interactions.

Implications for Myc function: Studies on the b-HLH and bZip regionswithin a number of eukaryotic transcription factors have distinguishedtwo essential yet separable functions for these domains: dimerization(HLH, Zip) and DNA-binding activity (basic region) (9, 10). Here we showthat the b-HLH-Zip domain of c-Myc is capable of specific interactionwith a newly identified b-HLH-Zip protein, Max. Our in vitro experimentsare consonant with studies on the structure and properties of Myc (1)and may serve as a basis for understanding the mechanism of Myc functionin vivo. Using anti-Myc, we have identified the Max protein inimmunocomplexes from avian and human cells, a further indication thatthe Myc-Max association is likely to be biologically relevant (16).

Two regions within the c-Myc protein appear to be critical for c-Mycfunction as judged by assays for cotransformation, inhibition ofdifferentiation, and suppression of endogenous Myc expression; these are(i) an approximately 40- to 60-amino acid segment centered about residue120, and (ii) the 95-amino acid carboxyl-terminal region (3-5). Ourresults suggest that the carboxyl terminus mediates association with Maxand formation of a sequence-specific DNA-binding complex. The mutationsthat negatively affected the ability of c-Myc to interact with Max andbind CM-1 (FIGS. 4 and 7A-G), such as deletion or disruption of thezipper, are either identical or very similar to those that abolish c-Mycactivity in biological assays (3-5).

The dimerization function that resides within the c-Myc HLH-Zip domainappears to be independent of the basic region, which is likely todirectly mediate DNA binding. The same c-Myc basic region deletionmutant that had no effect on association with Max completely abolishedthe ability of the Myc-Max complex to bind the CM-1 DNA probe (FIG. 7C).This basic region deletion mutation also abolished the ability of c-Mycto transform Rat 1 cells in collaboration with bcr-abl, while deletionof the adjacent upstream segment had no effect (16). Replacement of thec-Myc basic region with that of MyoD was likewise biologically inactivealthough, as expected, the chimeric protein was capable of associationwith Max (FIG. 4). Taken together, these results demonstrate that boththe dimerization and DNA-binding activities that reside in thecarboxyl-terminal bHLH-Zip domain are essential for important aspects ofc-Myc activity.

A striking finding of our study is that Max interacts specifically withthree members of the Myc family of proteins. Numerous attempts todemonstrate heterodimer formation between Myc and other b-HLH, bZip, andb-HLH-Zip proteins have not been successful (10, 15, 16). However, underour assay conditions Max is capable of associating with c-Myc, N-Myc,and L-Myc (FIG. 5A). Other proteins that contain related dimerizationdomains, including the b-HLH-Zip proteins USF and AP-4, did notassociate (FIG. 5B). Leucine zipper segments alone determine specificityin Fos-Jun association (51) and act to organize the two proteins in aparallel array (13). Max and the Myc proteins, however, all have HLHdomains in addition to zipper regions, and our data show that theintegrity of the HLH region is also important for heterodimer formation(FIG. 4). If an initial interaction between parallel zipper regions isrequired for proper orientation, then the appropriate alignment ofcontiguous HLH regions required for proper orientation might influencebinding. In Max and the Myc family, the hydrophobic residues of theputative leucine zipper appear to maintain their heptad spacing wellinto helix II, possibly extending the coiled-coil interaction. Bycontrast, in USF (47) the heptad phasing is disrupted at the helixII-zipper boundary, and in AP-4 (48) the hydrophobic array does notextend as far into helix II (FIGS. 3A-B). While it remains to bedetermined whether these differences are important for the apparentrestricted specificities of binding, other factors are likely toinfluence association, including the size and composition of the loopregion (11), the nature of specific residues within the helical segments(52), and the presence of other domains in the protein that mayfacilitate or block interaction. Although we have assumed that Myc andMax interact to form dimers, it is possible that they may alsoparticipate in higher order associations.

The fact that N-Myc and L-Myc as well as c-Myc specifically associatewith Max suggests that Max may serve to integrate the functions of thethese three proteins that are differentially expressed duringdevelopment, differentiation, and neoplasia(1). If so, Max might beexpected to be expressed in at least as many cell types as are Mycfamily proteins. Initial experiments with Northern (RNA) blottingindicate that a 2.1-kb Max RNA is expressed in many cells and tissues atconcentrations comparable to those of c-Myc. In addition, low stringencySouthern (DNA) blot analysis suggests that Max is highly conserved as asingle gene or a small family of genes in vertebrate genomic DNA, but isabsence from invertebrates that also lack Myc homologs(16). Theseresults are consistent with the possibility that Max, or a small numberof Max-related proteins, interacts with Myc family proteins to mediatetheir specific biological functions. Whether Max can also beoncogenically activated poses an interesting biological question.

Important questions raised by this work concern the way in which theproperties of Myc and Max are altered through association. Ourexperiments demonstrate that complex formation generatessequence-specific DNA binding activity for the CM-1 oligonucleotideunder conditions where neither Myc nor Max alone bound significantamounts of probe (FIG. 7). This oligonucleotide contains the CACGTGconsensus, which serves as a binding site for presumptive Myc homodimers(17,36). That this is a weak binding site may be reflected by the lowbut detectable binding by in vitro translated Myc. No binding by Maxalone could be detected, indicating that either Max doe not recognizeCM-1 or that it does not homodimerize under the conditions of the assay.A key point becomes whether the Myc-Max heterocomplex has a specifityfor DNA binding that is distinct from that of either of the homodimers.By analogy with MyoD and the E2A proteins, each member of a Myc-Maxcomplex might contribute half-site recognition in defining DNA-bindingspecificity (38). The Myc-Max complex can be used directly to select aputative new binding sequence with the method for preferential bindingand amplification of random sequences (38).

Another major question concerns the function of the Myc-Max complex. Ithas been suggested that Myc may function in transcription, DNAreplication, or both (1). The characteristics of the Myc-Max complexplaces there proteins in the same general class as b-HLH transcriptionfactors, but the results do not rule out other possible functions. Theessential amino-terminal region of c-Myc has been shown to act as atranscriptional activation domain when linked to yeast or prokaryoticDNA-binding domains (53). However, introduction of c-myc alone intocells only induces variable, usually low, activation of differentpromoters (54). While the HLH-Zip region constitutes the very carboxylterminus of all the Myc family members, the HLH-Zip region of Max isnearly 50 residues from its carboxyl terminus (FIG. 3C). This region,which probably extends past the dimerized regions of Myc and Max,contains additional acidic and basic patches (FIGS. 2C-D) that couldinteract with components of the transcriptional machinery or otherfactors (55). Whatever their function, the ability of these polypeptidesto form multiprotein complexes suggests that the differential regulationof their relative concentrations could be important determination ofMax-Myc family associations, consequent DNA-binding specificities, andultimately, the influence of Myc on cell proliferation and behavior.

SECOND SERIES OF EXAMPLES

Here we have identified the Myc-binding protein Max in vivo and haveshown that Myc and Max are associated in the cell.

The protein encoded by the c-, L- and N-myc protooncogenes areshort-lived nuclear phosphoproteins which possess DNA binding andprotein dimerization domains structurally related to those found in anincreasing number of transcription factors (see 56-58). For this classof factors dimerization is mediated by a putative helix-loop-helixregion which in some cases (as in the Myc family proteins) is contiguouswith a leucine zipper motif (HLH-Zip). Dimerization is required forspecific DNA binding by the short stretch of basic amino acids (b) whichprecedes the HLH-Zip region. (see 58, 59 for reviews) As describedabove, by employing a functional cloning strategy we previouslyidentified a novel human cDNA which encodes a bHLH-Zip protein Max. Maxassociates in vitro with the c-Myc, N-Myc, and L-Myc proteins but notwith other bHLH-Zip proteins tested (60). A murine homolog of Max hasalso been identified (61). Association between Myc and Max requires theHLH-Zip regions of both proteins (60, 61). In addition, the humanc-Myc:Max complex binds to DNA in a sequence-specific manner underconditions where Myc or Max alone display relatively, weak binding. DNAbinding is dependent on the basic region as well as the HLH-Zip domainsof both partners (60-62). Given the results of these in vitro studies itseemed important to identify and characterize Max and determine whetherit associates with Myc in vivo.

EXAMPLE 5 Myc Associates in vivo with Max

To study Max in vivo we produced an antiserum against a purified fusionprotein containing the 124 carboxyl-terminal residues of human Maxlinked to the carboxyl-terminus of glutathione-S-transferase(GST-MaxCI24). Specifically, GST-Max C124 was constructed and purifiedas described above. Affinity purified antibodies to the 12carboxy-terminal amino acids of human c-Myc (anti-Myc) have beencharacterized elsewhere (63).

The anti-GST-MaxCI24 serum (anti-Max) was used to immunoprecipitate maxfrom whose cell lysates prepared from [³⁵ S]-methionine-labeled humanBurkitt's lymphoma cells (Manca). Specifically, immunoprecipitationsfrom from [³⁵ S]methionine-labeled cells were performed using highstringency conditions as previously described [B. Luscher, L. Brizuela,D. Beach, R. N. Eisenman, EMBO J. 10, 865 (1991)]. All SDS PAGE sampleswere resolved on 15% acrylamide gels under reducing conditions. Fortwo-dimensional tryptic peptide analysis, Max proteins wereimmunoprecipitated and treated with alkaline phosphatase prior to gelpurification and peptide mapping (64).

SDS-PAGE analysis of an anti-Max immunoprecipitate revealed apredominant doublet with relative molecular masses of 21,000 and 22,000(M_(r) 21K and 22K) which was not recognized by the cognate preimmuneserum (FIG. 8A). Immunoprecipitation of the 21/22K proteins could becompetitively inhibited by excess GST-MaxC124 protein, but not by excessGST alone, suggesting that p21/22 are recognized through determinantsspecific to the Max segment of the immunogen. To determine whetherp21/22 are also structurally related to Max we compared two-dimensional³⁵ S-methionine tryptic peptide maps of the protein generated by invitro transcription/translation of the p21 Max cDNA clone and of thep21/22 proteins from Manca cells. FIGS. 8B-E show that the labeledpeptide patterns are superimposable suggesting that the p21/22 proteinsrecognized by anti-Max are highly related to Max.

As both p21 and p22 proteins can be identified in Manca as well as othercell types (FIG. 8A) (65) it was important to determine the relationshipbetween the two proteins. They did not appear to be differentiallyphosphorylated forms of the same protein since phosphatase treatment didnot resolve the p21/p22 doublet into a single species (65). Previouswork had identified two Max cDNAs differing only by the addition of a9-amino acid segment N-terminal to the basic region (60,61), In vitrotranslation of the two variant cDNAs shows that they differ in Mr byapproximately 1K and that their individual electrophoretic mobilitiescorrespond to those of p21 and p22 immunoprecipitated from Manca cellswith anti-Max (FIG. 8F). These data suggest that p21 and p22 are Maxproteins which differ by the 9-amino acid insertion. [The nine aminoacid insertion would not be expected to contribute to the trypticpeptide pattern shown in FIG. 8B since the initiating N-terminal [³⁵S]methionine of Max is likely to be removed (R. Moerschell, S. Hosokawa,S. Tsunasawa, F. Sherman J. Biol. Chem. 265, 19639 (1990).] We conclude,on the basis of antigenicity, electrophoretic mobility, andtwo-dimensional peptide mapping analysis that p21 and p22 are encoded bymax.

Referring to FIG. 8A in more detail, Max protein was immunoprecipitatedfrom [³⁵ S]methionine labeled Manca cells using anti-GST-MaxC124(α-Max). Preimmune serum (Pre-imm) served as a background control, whileexcess immunogen (GST or GST-Max) was used to compete for specificanti-Max binding. Referring to FIGS. 8B-E, the left-hand panel showsSDS-PAGE analysis of the immunoprecipitated and in vitro translatedproteins used for peptide maps of [³⁵ S]methionine labeled proteincomparing in vitro translated p21 Max (IVT) with in vivo labeled proteincomparing in vitro translated p21 Max (IVT) with in vivo labeled p21/22Max proteins (Manca). Referring to FIG. 8C, the two Max cDNA's (IVT p21and p22) were translated in a reticulocyte lysate and compared in2-dimensional SDS-PAGE with in vivo labeled Max polypeptides (in vivoα-Max).

Proteins belonging to the Myc family have long been characterized asnuclear phosphoproteins (see ref. 66). The Max is also nuclear can bedemonstrated by immunofluorescence analysis of fixed HeLa cells.Specifically, indirect immunofluorescence staining was performed onfixed HeLa cells using previously described methods [D. K. Palmer and R.L. Margolis, Mol. Cell Biol. 5, 173 (1985)]. Briefly, cells were fixedwith paraformaldehyde, permeabilized with Triton X-100, blocked withbovine serum albumin and incubated with affinity purified anti-Max orpolyclonal and anti-Myc serum. Secondary antibody wasfluorescein-conjugated goat anti-rabbit immunoglobulin.

Anti-Max produces granular nuclear staining exclusive of nucleoli, asobserved for Myc (FIGS. 9A-H) (67). In addition both the p21 and p22 Maxproteins appear to be predominantly nuclear as shown by cellfractionation experiments(65). That Max is a phsophoprotein wasdemonstrated by immunoprecipitation of radioactive p21/p22 from [³² PO₄]-labeled Manca cells (FIG. 8B). [In vivo [³² P]orthophosphate labelingas well as in vitro CKII kinase assays were performed as previouslydescribed (64).] Several major in vivo phosphorylation sites on c-Mychave been shown to correspond to those phosphorylated by casein kinaseII (CKII) in vitro (64). Because Max also contains putative CKIIconsensus phosphorylation sites we determined whether CKII wouldphosphorylate Max in vitro by treating immunoprecipitated p21/p22 Maxwith purified CKII and γ-³² P-ATP. FIG. 8B shows that radiolabeledphosphate was specifically incorporated into both Max proteins. Trypticphosphopeptide maps of in vivo [³² PO₄ ]-labeled Max are identical tothose produced by CKII phosphorylation in vitro (65). Thus both Max andMyc proteins appear to be in vivo targets for CKII phosphorylation.

Referring to FIGS. 9A-H in more detail, subcellular localization of Maxprotein was assayed by indirect immunofluorescence on fixed Hela cells.Anti-Max and polyclonal anti-Myc immunoreactive proteins were detectedwith FITC-labeled goat-anti-rabbit Ig secondary reagent. Excessimmunogen (block) or preimmune serum (Prei.) were used as negativecontrols, respectively. Phase-contrast images of the immunostained cellsare shown to the right. Referring to FIG. 9I, Max polypeptides wereimmunoprecipitated from [³² P]orthophosphate labeled cells (α-Max³² Pi)or from unlabeled cells and phosphorylated in vitro with casein kinaseII (CKII, α-Max). The immunogen (GSTMaxC124) served as an excellentsubstrate for CKII when added as a blocking reagent (b).Autophosphorylation of the β subunit of CKII ("-") is shown in theenzyme only control.

Myc proteins have extraordinary short half-lives, on the order of 20-30minutes (63,68). In contrast both Max proteins are highly stable asdemonstrated by the pulse-chase analysis shown in FIG. 10. No change inthe levels of pulse-labeled Max are detectable 6 hours after removal oflabel, and Max appears stable even after a 24-hour chase period (65). Inaddition the relative levels of p21 and p22 Max are unaltered during thecase period, consistent with the idea that the two proteins areindependent translation products (FIG. 10).

Referring to FIG. 10, to analyze protein stability, K562 cells werepulse-labeled with [³² S]methionine for 30 min (P), then "chased" forvarious lengths of time in the presence of excess nonradioactivemethionine. Samples were immunoprecipitated under high stringencyconditions with anti-Max and subjected to SDS PAGE. GST-MaxC124 was usedto block specific immunoprecipitation (b).

The c-myc gene belongs to the class of immediate early genes in thatwhile myc RNA and protein are virtually undetectable in quiescent cellsthey are transiently induced to high levels which several hoursfollowing mitogenic stimulation (69, 70). The peak of c-myc expressionis followed by a decrease to a basal level that remains invariantthroughout the cell cycle (71, 72, 73). Since c-myc and max areexpressed in many of the same cell types (70), we asked whether max wasalso an immediate early gene by examining its expression levelsfollowing mitogen stimulation of serum-starved A31 Balb/c 3T3 cells.Specifically, quiescent A31 Balb/c-3t3 cells were serum stimulated aspreviously described [M. E. Greenburg and E. B. Ziff, Nature 311, 433(1984)]. [³ H]thymidine incorporation was measured in triplicate from 24well plates as described [D. F. Bowen-Pope and R. Ross, J. Biol. Chem.257, 5161 (1982)]. In parallel, RNA was extracted by theacid-guanidinium thiocyanatephenol-chloroform procedure [P. Chomczynskiand N. Saachi, Anal. Biochem. 162, 156 (1987)]. For Northern analysis,10 μg of total RNA was hybridized with a 560 bp Max probe. For analysisof steady state protein levels, Max polypeptides were immunoprecipitatedfrom unlabeled cultures (3×10⁶ cells), blotted to nitrocellulose andreprobed with anti-Max and [¹²⁵ I]Protein A.

Steady state expression levels were determined by immunoblotting withanti-Max and by Northern blotting. Surprisingly both Max protein and maxRNA were readily detected in quiescent culture (FIGS. 11A-C). Additionof serum clearly resulted in entry of cells into G1 and S phases, asjudged by the 20 fold increase in 3H-TdR incorporation (FIGS. 11A-C) andan early increase in c-myc RNA (65). However no significant change insteady state max RNA or protein levels was observed. In addition Maxexpression levels are also not altered during the cell cycle asdetermined by centrifugal elutriation experiments(65). Therefore Max isnot a member of the class of mitogen inducible genes and its levels ofexpression are independent of cell growth.

Referring to FIGS. 11A-C, A31 Balb/C 3T3 cultures were serum depletedfor five days prior to stimulation with 15% fetal calf serum. [³H]thymidine incorporation was measured in 2 hour pulse labelingsfollowing addition of serum (top). Max mRNA levels were analyzed byNorthern blotting using the max cDNA as probe (60). An ethidiumbromide-stained agarose gel was used to normalize the amount of RNA (10μg, middle). To monitor steady state levels of Max protein, anti-Maximmunoprecipitates (from unlabeled cells) were resolved by SDS-PAGE,transferred to nitrocellulose and reprobed with anti-Max and [¹²⁵I]-Protein a (bottom). Anti-Max antibody serves as a control for ]¹²⁵I]-Protein A-reactive immunoglobulin (Ab). The time course is in minutes(') or hours following serum stimulation.

In vitro c-Myc homodimerizes poorly, if at all (62, 74), while Maxself-associates, but preferentially forms heterodimers with c-Myc (60).A major question raised by the in vitro demonstration of Myc:Maxassociation is whether these proteins also interact in vivo. To answerthis question we began by examining the conditions required forimmunoprecipitation of Myc from cells. Analyses of Myc proteins byimmunoprecipitation are frequency carried out using a mixture ofnon-ionic and ionic detergents which permit efficient extraction of Mycfrom nuclei and decrease non-specific binding of proteins to theimmunocomplex (67, 75-78). As deoxycholate and SDS, however, disrupt theMyc:Max complex formed following in vitro translation of both proteins(65), in vivo association between Myc and Max might be similarlydisrupted under standard "high stringency" immunoprecipitationconditions. Standard "high stringency" (HS) immunoprecipitations werecarried out as previously described (76). Briefly, cells were lysed inAb buffer (20 mM Tris-HCI, pH 7.4, 50 mM NaCl, 1 mM EDTA, 0.5% NP40,0.5% deoxycholate, 0.5% SDS, 0.5% aprotinin), sonicated, clarified bycentrifugation and subjected to immunoprecipitation with saturatingamounts of antibody. Immunocomplexes were collected using proteinA-Sepharose CL4B (Sigma). The beads were washed sequentially with RIPAbuffer twice (10 mM Tris. pH 7.4, 0.15 M Nacl, 1% NP40, 1% deoxycholate,0.1% SDS, 0.5% aprotinin), with high salt buffer (2M NaCl, 10 mM Tris,pH 7.4, 1% NP40, 0.5 % deoxycholate) and finally with RIPA buffer.

However, the Myc:Max oligomers formed in vitro were not disrupted inbuffers containing only non-ionic detergents ("low stringency"conditions) (60). FIG. 12A shows a comparison of anti-c-Myc and anti-Maximmunoprecipitates from Manca cells carried out under high and lowstringency conditions. To increase the specific activity of the Maxpolypeptides for low stringency (LS) immunoprecipitations, cells weremetabolically radiolabeled for 1 hour. All subsequent steps were done at4° C. to stabilize Myc:Max complexes. Washed cells were lysed in PBScontaining 1% NP-40 and a cocktail of protease and phosphataseinhibitors (0.2 mM phenylemthylsulfonyl fluoride, 0.7 μg/ml pepstatin,0.5% aprotinin, 10 mM NaF, 50 mM β-glycerophosphate). The lysate (1×10⁷cells/ml) was sonicated on ice and microfuged to clarify. 5×10⁶ cellequivalents were immunoprecipitated with 5 μg of anti-Max and collectedon Protein A-Sepharose beads. Low stringency buffer was used to wash theprecipitate four times including a single wash with 0.5M NaCl. Thenonimmunoreactive component of the complex was eluted with 0.5 ml Abbuffer (described above), and precipitated under high stringencyconditions. Samples were analyzed by SDS-PAGE under reducing conditions.

The immunocomplexes formed using high stringency buffer contain eitherp64 c-Myc or p21/22 Max (FIG. 12A, lanes HS). Reduction of thestringency of the buffer results in an increase in the backgroundprecipitation as well as the appearance p21/22 in the anti-Mycprecipitate, and p64 in the anti-Max precipitate. (FIG. 12A, comparelanes LS). That these proteins are specifically precipitated isdemonstrated by the ability of Max and Myc immunogens to competitivelyblock their precipitation (FIG. 12A, compare lanes b and lanes LS),while the elevated background is unaffected. Furthermore, FIG. 12A,(α-Myc lanes) shows that anti-Myc can cleanly precipitate Myc proteinreleased from a low stringency anti-Max immunocomplex by addition of SDSand deoxycholate. Similarly, anti-Max can precipitate Max proteinreleased from the anti-Myc complex in the same manner (FIG. 12A, α-Maxlanes). The ability of the anti-Myc and anti-Max sera to precipitate thecomplex without disrupting it is consistent with our results on Myc:Maxoligomers formed in vitro (60). That Max and Myc can becoimmunoprecipitated from cells under nondisassociating conditionssuggests that these two proteins do interact in vivo. Similarly, we candemonstrate association between N-Myc and Max as well as betweendifferent retrovirally encoded v-Myc proteins and Max (65).

Considering the highly stable nature of Max it was of interest todetermine whether the short-half life of c-Myc might be affected by itsassociation with Max. Myc protein stability was evaluated by pulse-chaseexperiments using BK3A cells, a chicken bursal lymphoma cell line inwhich Myc protein metabolism has been extensively studied (68).Following the pulse label, and at different time points during the"chase" period, the cells were lysed under low stringency conditions andMyc proteins immunoprecitated under high stringency conditions withanti-Myc to determine the total amount of radiolabeled Myc present (FIG.12B). Specifically, for analysis of protein and complex stability, cellswere pulse-labeled with [³⁵ S]methionine for 30 min, washed free ofunmetabolized radiolabel, and chased in the presence of excess coldmethionine (0.5 mM). At specified time points, lysates were prepared inlow stringency buffer, immunoprecipitated under either high or lowstringency conditions and analyzed by SDS PAGE.

In parallel, the amount of labeled Myc protein associated with Max wasdetermined by immunoprecipitation with anti-Max under the low stringencyconditions (FIG. 12C). Although Myc protein can be resolved under theseconditions we verified the amount of Myc present in the Max complex bytreating the a-Max immunocomplex with high stringency buffer followed byreimmunoprecipitation with anti-Myc (FIG. 12D). The results clearly showthat the majority of the newly synthesized Myc protein is present in thecomplex with Max and, furthermore, that Myc's half-life is unchanged byits association with Max. It is important to bear in mind however thatthese low stringency lysis conditions may not efficiently extract all ofthe Myc protein (65).

Referring to FIG. 12A in more detail, [³⁵ S]methionine labeled Mancacell lysates were immunoprecipitated under the described high (HS) orlow (LS) stringency detergent conditions. An excess of the cognateimmunogen was used to block specific immunoprecipitation (b). To verifythe identity of the coprecipitated component, low stringency Myc:Maxcomplexes were disassociated with SDS and reimmunoprecipitated underhigh stringency conditions with the converse antiserum (α-Myc or α-Max).Referring to FIGS. 12B, the stability of Myc protein was analysed bypulse-chase labeling (30 min. pulse label) of BK3A, an avian bursallymphoma cell line. Low stringency extracts of Myc protein were directlyimmunoprecipitated under high stringency conditions (HS α-Myc) orcoprecipitated in a complex with Max (LS α-Max). To verify the levels ofMyc protein found in anti-Max immunoprecipitates, low stringencycomplexes were disassociated with ionic detergent and reprecipitatedwith anti-Myc (LS α-Max→HS α-Myc). Identical exposures are shown.

Here we have identified the Myc-binding protein Max in vivo and haveshown that Myc and Max are associated in the cell. Most if not all ofthe newly synthesized Myc passes through an in vivo complex with Max: aresult consistent with the dimerization properties of Myc and Maxobserved in vitro (60, 62, 74). The half-life of Myc protein is notaltered by its association with Max although Max itself is an extremelystable nuclear phosphoprotein.

Max protein and RNA are readily detected in quiescent cells at levelsthat are unchanged by serum stimulation or cell cycle phase. However inresponse to mitogenic stimulation Myc levels increase from nearbackground in quiescent cells to a peak of expression which thendeclines to a basal level prior to S phase (69). During the cell cycleMyc synthesis and rapid turnover are maintained at a constant basallevel (71, 72, 73). Therefore in contrast to Myc, Max expression appearsto be independent of the growth state of the cell.

The contrasting properties of Myc and Max suggest first, that it is theabundance of Myc which limits or drives formation of heterocomplexes,and second that Myc function is mediated through a Myc:Maxheterocomplex. Short-lived Myc monomers may be continually competingwith Max homodimers for interaction with Max. One possibility is thatMax homodimers function in a manner distinct from Myc:Mas heterodimersand that Myc serves to transiently "switch" Max between its differentactivities. Thus the extraordinary degree to which Myc expression isregulated, and the loss of this regulation during oncogenic activation(see 78), may critically influence the balance hereto and homodimerfunction. It will be of interest to determine whether complex formationof function is further regulated by CKII phosphorylation, the expressionpattern of two alternative Max proteins, or potential interactions withother cellular components.

In retrospect, the experiments described in the prior art lead to apicture of Max as a highly conserved, stable nuclear phosphoproteinexpressed in many cell types. Although numerous immunoprecipitationexperiments have been carried out over the last five years with anti-Mycantibodies, Max had never been previously identified as being associatedwith Myc. One explanation may be related to the immunoprecipitationconditions used to study Myc, which are usually high stringency (i.e.,with multiple detergents) in order to efficiently extract Myc fromnuclei and reduce background. To determine whether Myc and Max interactin vivo we carried out immunoprecipitations with either anti-Myc oranti-Max antibodies under low stringency conditions. Such conditionsresult in an increase in background, but among the proteins precipitatedMyc and Max are visible. Their presence was confirmed by solubilizingthe immunocomplexes using high stringency buffer followed by a secondimmunoprecipitation with anti-Myc and anti-Max. The results clearlydemonstrate that both Myc and Max are present in anti-Max and anti-Mycimmunocomplexes.

Our experiments show that Myc and Max are likely to associate in vivoand that a significant fraction of the population of each protein isinvolved in the interaction. These findings raise a number of questionsrelevant to the physiological function of Myc. Since Max appears toself-associate (60), while Myc homodimerizes weakly if at all, it ispossible that populations of relatively stable Max homodimers andunstable Myc:Max heterodimers exist in cells. Max or Myc alone have beenshown to bind the sequence CACGTG, presumably as homodimers (62). Recentexperiments indicate that Myc:Max heterodimers bind this sequence morestrongly (60) and that the binding specificity is unchanged (K.Blackwell and E. Blackwood, unpublished data). Even through homodimersand heterodimers might bind the same sequence, the difference in bindingstrength, as well as the very nature of the complex bound to DNA (i.e.,the heterocomplex obviously has a structure quite distinct from thehomodimeric complex), could have profound consequences. Whatever theseeffects may be they are probably largely dependent on the levels of Mycprotein. This is because Myc may be competing for binding with Max:Maxhomodimers and because Myc is degraded so rapidly while Max is highlystable. Thus, even small changes in Myc regulation at transcriptional orpost-transcriptional levels might be biologically important by affectingthe concentration of heterodimeric complexes.

If we assume that Myc exerts its proliferation promoting functionthrough its interaction with Max then events that interfere with thisinteraction are likely to modulate Myc's function. This would includepost-translational alterations, such as phosphorylation, which mightafter Myc's ability to associate with Max, or the presence of otherproteins interacting with Myc, Max, or both. Such proteins acting at thelevel of complex formation might act as tumor suppressor proteins.Alternatively, such proteins could also function after the complex isformed: to attenuate its activity or prevent its interaction with otherpositively acting factors. Finally, the possibility that Max is itself anegative regulator of growth needs to be considered. One scenario isthat Myc diverts Max's proliferation suppressor activity into aproliferation promoting activity by forming an active complex with it.The finding that Myc and Max can interact specifically both in vitro andin vivo now permits a direct test of these ideas.

Constitutive high-level myc expression has previously been shown to leadto changes in growth rate (79, 80), increased sensitivity to growthfactors (81-83), inhibition of differentiation of numerous cell types(84-87), and the capacity to cooperate with other activated oncogenes inthe tumorigenic conversion of normal cells (88-90; for review, see 91).Thus, it is not difficult to rationalize even subtle changes in mycregulation with an increased potential for neoplastic growth.

The findings that c-Myc has sequence-specific DNA-binding activity andis also capable of specific association with another HLH-Zip proteinputs it firmly in the same general functional class as the bHLH and bZiptranscription factors. Nonetheless, Myc would seem to be structurallydistinct from these other proteins, in that it possesses contiguous HLHand zipper domains and appears to have its own restricted set ofinteractions. Whether this restricted set includes the other HLH-Zipproteins remains to be determined. We surmise that both Myc:Myc andMyc:Max complexes function in transcription activation or repressionthrough their specific DNA-binding activities, and that formation ofcomplexes is dependent on the relative levels of expression of myc andmax genes. This model does not exclude a role for Myc in DNAreplication, since its transcriptional activity could well affect theexpression of genes involved in induction of S phase. Alternatively, Mycmight be more directly involved in DNA synthesis through binding to theclass of replication origins that contain enhancer or promoter elements(see 92). The delineation of the specific interactions of Myc mayprovide a means to resolve these possibilities and elucidate the role ofMyc in normal and neoplastic cell behavior.

THIRD SERIES OF EXAMPLES

Here we have identified the Max-binding protein Mad in vitro and haveshown that Mad can compete binding of Myc to Max.

The Materials and Methods and Discussion sections for the Third Seriesof Examples appears at the end of EXAMPLE 11.

EXAMPLE 6 Identification of a New Max Binding Partner

There are several findings that suggest the potential for a protein,other than Max, that could regulate Myc activity: Myc expression isregulated during entry to and exit form the cell cycle while Maxexpression is constitutive, vast differences in the stability of the twoproteins and differences in the cell type and tissue distribution. Onemight speculate that a protein that could alter the activity or theavailability of Max might influence Myc function. Therefore, we haveused purified Max protein to screen an λgt11 expression library for Maxbinding partners. Max protein was overexpressed using the baculovirusexpression system. Sf9 cells were infected with a recombinantbaculovirus containing the Max cDNA insert and Max protein was purifiedto apparent homogeneity from a cytoplasmic cell extract using cation andanion exchange chromatography. We tested this protein preparation forMax DNA binding activity using the electrophoretic mobility shift assay[EMSA] (FIG. 13A).

The purified Max preparation formed a retained complex in the presencethe Myc/Max binding site, CM-1. This complex was a result of Max bindingbecause it is further retained in the gel matrix in the presence of ananti-peptide antibody specific for Max. The binding activity in the Maxpreparation was specific for the CM-1 oligo as it was competed byincreasing amounts of unlabeled CM-1 but not by equivalent amounts of aunrelated oligo (FIG. 13B).

The purified Max was also able to form heterodimers with Myc and wastherefore competent for dimerization as well as DNA binding (data notshown). We concluded that the insect cell expressed Max protein washighly active in DNA binding and heterodimerization and thereforesuitable to use as a probe to screen for potential max binding partners.

Max was first identified by screening an expression library was aniodinated fusion protein containing the C-terminal 92 amino acids of Myc(FIRST SERIES OF EXAMPLES, above). We have used a similar approach toscreen for Max binding partners however, we have used casein kinase II(CKII) and [γ-³² P]ATP to label Max. The Max protein has two CK IIphosphorylation sites in the n-terminus (serines 2 and 11) and a clusterof 5 serine residues near the C-terminus of the molecule. Max proteinfrom insect cells is partially phosphorylated but can still be labeledto high specific activity by CKII in vitro (data not shown).Furthermore, the β-subunit of CKII is known to be autophosphorylated butwas not labeled under our reaction conditions and therefore Max was theonly labeled protein in the probe preparation (data not shown). Thelabeled Max was used to screen a λgt11 expression library constructedfrom the baboon pre-B cell line 594-S. In the initial screen 10⁶individual phage were plated and screened. Labeled Max bound to abacterial lysate infected with a phage (Max 14) that encoded Max (above)(FIG. 13C).

FIGS. 13A-C Identification of Max binding partners. The ability of maxpurified from Sf9 cells to bind the CM-1 binding site was assayed by theelectrophoretic mobility shift assay (FIG. 13A and 13B).

FIGS. 13A shows Max DNA binding assayed in the absence (-) or thepresence of a Max specific anti-peptide antiserum (αMax). αMax+blockindicates the inclusion of the immunizing peptide in the bindingreaction.

FIGS. 13B shows Max binding activity assayed in the presence ofunlabeled CM-I or an unrelated oligonucleotide, MREA. The amount ofcompeting oligonucleotide is give in ng; "-" denotes no unlabeledoligonucleotide in the binding reaction. The position of the free probeand the Max homodimer mobility shift is as marked. The asterisk denotesthe antibody:Max:Max complex.

FIG. 13C shows ³² P-labeled Max binding to a filter containing phagelysates from different gt11 lambda clones. Max 14 wash previouslyidentified as a binding partner for Myc. λ1 encoded a lacZ fusionprotein with no specific Max binding activity and served as a negativecontrol in this experiment.. λ10, 11, and 26 encoded lacZ fused topotential Max binding partners.

Three additional phage encoded proteins that were positive for Maxbinding through multiple rounds of screening and purification (FIG.13C). It was likely that these clones encoded Max or one of the Mycfamily members fused to lacZ. λ10 and 11 hybridized to a Max DNA probeand encoded a protein that was immunologically related to Max. λ26 wasnot related to any of the Myc family members by DNA hybridization andtherefore represented a potentially new binding partner of Max. We havesubcloned the cDNA insert from λ26 termed it Mad-1 (Max associateddimerization). The baboon Mad-1 cDNA had a 186 amino acid open readingframe fused to lacZ. Using this partial baboon Mad-1 cDNA from λ26 wehave isolated a human Mad-1 cDNA from a embryonic lung cDNA library(FIGS. 14A-B).

FIGS. 14A-B Nucleotide and amino acid sequence of the human Mad-1 cDNA.The nucleotide and the amino acid sequence of the coding region of the3.2 kb human Mad1 cDNA from the WI26 λgt10 library is shown. Nucleotidepositions are indicated. Amino acid positions are denoted by bold facednumbers and in frame stop codons are shown. The basic region homology isboxed and the positions of the positively charged residues in thisregion are marked by +. The shaded boxes locate helix I and helix II.The amino acids that form the hydrophobic heptad repeat are given inbold underline text. The region rich in acidic amino acids is locatedbetween amino acids 152 and 189.

Comparison of the baboon and the human CDNA sequences revealed no aminoacid differences. We believe that the human cDNA is full length becausethe first AUG encoded in the RNA is in good translational context and isproceeded by an in frame amber stop codon. In addition, the human Mad-1cDNA is 3.2 kb in size in agreement with the size of the Mad-1 RNA asdetermined by northern blotting(data not shown).

The Mad-1 cDNA encodes a protein of 221 amino acids with a predictedmolecular weight of 25,200 daltons. A search of the protein data basewith the predicated Mad-1 amino acid sequence revealed no identitiessuggesting that it is a previously unrecognized protein. However, theMad-1 protein sequence is a near perfect match to the consensus sequencedetermined for the basic-helix-loop-helix (b-HLH) family oftranscription factors (FIGS. 15A-B).

Mad-1 is most similar to the Drosophila proteins extramacrocheatae andhairy had has some similarity to the Myc family members. The similarityto the Myc proteins is limited to the basic region and suggests thatMad-1 is not another member of the Myc family. Mad-1 has littlesimilarity outside the conserved amino acids of the b-HLH consensus toMax or two other b-HLH proteins (USF and TFE3) that bind the CACGTGelement. Like Myc and Max, Mad-1 has a heptad repeat of hydrophobicamino acids following helix II of the HLH domain. This repeat of aminoacids might form a structure similar to the coiled-coil leucine zipperdomain that has been shown to mediate dimerization other transcriptionfactors with similar domains. The b-HLH-zipper structure of Mad-1 is asimilar to those found on Max and Myc but the structural organization ofthe three proteins is quite different (FIG. 15 C).

FIGS. 15A-C. Comparison of Mad-1 to other b-HLH proteins. The predicatedamino acid sequence of Mad-1 is compared to other members of the b-HLHfamily of transcription factors and to the b-HLH consensus (FIGS.15A-B). The amino acids are denoted by the single letter code. TheDrosophila proteins EMC (extramacrocheatae) and hairy were found to bemost similar to Mad-1 is searches of the data base while TFE3 and USFboth recognize the same DNA binding site (CACGTG) as Myc and Max. Thematches to the b-HLH consensus are shaded and the residues that form aheptad repeat of hydrophobic amino acids are shaded and boxed. Theprimary structure of a generalized b-HLH-zipper protein is shownschematically at the bottom of the panel. The structural organization ofMad-1, Max and Myc is shown in FIG. 15C. The numbers indicate amino acidposition. The basic region, helix-loop-helix, and leucine zipperhomologies are as indicated.

In Mad-1, the b-HLH-zipper region is localized to the middle of theprimary amino acid sequence. By contrast, the b-HLH-zipper domains ofMyc and Max are localized to the carboxy and the amino termini,respectively. It is not clear if the different structural organizationof these proteins has functional consequences.

Myc and Max are both nuclear phosphoproteins. The similarity of Mad-1 tothese proteins suggests that it should be localized to the nucleus aswell. There is a potential bipartite nuclear localization (Dingwall andLaskey, (1991) signal in Mad-1 found between amino acids 20 and 50. Mycand Max are in vivo and in vitro substrates for casein kinase 11 (CKII).The COOH-terminus of Mad- I is rich in negative charge (amino acids152-189) and contains several potential CKII phosphorylation sites. Thisregion may function as a transcriptional activation domain similar theacidic region of Myc and other transcription factors. If utilized, CKIIphosphorylation of this region would increase the negative chargedensity of this region even further.

EXAMPLE 7 Mad-1 Binds Specifically to Max

In Order to test the binding specificity of Mad-1 we have constructed afusion protein between the COOH-terminal 186 amino acids encoded by thebaboon Mad-1 cDNA and glutathione-s-transferase. This fusion protein isexpected to contain the domains of the Mad- 1 protein requiredheterodimer formation and DNA binding. Either the purified fusionprotein (GST-Mad) or purified glutathione-s-transferase (GST) was addedto in vitro translation reactions programmed with RNAs encoding eitherMax or Max 9. Following translation the products were allowed to bind toglutathione-sepharose. The beads were washed extensively and the boundproteins were analyzed on SDS-PAGE gels (FIG. 16A).

Both Max and Max 9 were retained on the glutathione beads if they weretranslated in the presence of GST-Mad; however, neither protein wasretained in they were translated in the presence of GST alone. Thisindicated that the Max proteins were retained on glutathione beadsthrough the Mad segment of the fusion protein. If the Mad-1 cDNA wastranslated and assayed in a similar manner there was high backgroundbinding to GST alone; however, there was no Mad-1 binding to GST-Madabove this background level (data not shown). This suggested that Mad-1formed homodimers poorly. Because Mad1 seemed to bind theglutathione-sepherose non-specifically we were concerned that it mayhave a generalized non-specific binding activity. We, therefore, havedetermined which regions of Max are needed for interaction with GST-Mad.Various Max mutants were tested for binding to GST-Mad or GST (FIG.16B).

Proteins that had the basic region either Max or Max 9 deleted bothbound to GST-Mad. By contrast, deletion of the last two leucines fromthe Max hydrophobic heptad repeat abolished binding to GST-Mad (i.e.,leu₈₆ and leu₉₃ ; FIGS. 2C-D). These two leucines were also required forMax heterodimerization with Myc. These data suggest that the heptadrepeats of hydrophobic amino acids in Mad-I and Max are responsiblemediating their interaction. We have not tested the role of the HLHmotif in Mad-1:Max interaction but based on mutational analysis of othermembers of the b-HLH family the HLH region is expected to play a role indimerization as well.

Because Mad-1 and Myc both bind to max it seemed reasonable that Myc andMad-1 might also interact. Using conditions where we readily detectedMax interaction with GST-Mad there was no binding of C- or N-Myc toGST-Mad was detected above background (FIG. 16C).

Therefore, these results suggest that Mad-1 has dimerization propertiessimilar to those of Myc. Both Mad-1 and Myc homodimerize poorly but bothreadily form heterodimers with Max. It is likely that the physicalcharacteristics of Mad-1 and Myc that prevent their homodimerizationalso prevent their heterodimerization. We have also examined thegenerality of Mad-1 binding by testing other members of the bHLH familyand other proteins might be involved in Myc/Max function for interactionwith GST-Mad. USF, TFE3 and AP-4 all have a structure similar to Myc,Max and Mad (i.e. b-HLH-zipper motif) and USF and TFE3 have similar DNAbinding specificity to Myc and Max(i.e. bind the CACGTG site). MyoD hasonly a b-HLH motif whereas Fos and Jun use a leucine zipper fordimerization but use a region different from the basic region for DNAbinding. Rb and TFIID have been reported to be in vitro binding partnersfor Myc. None of these proteins were able to interact with GST-Mad abovebackground levels (FIGS. 16D,E,F).

FIG. 16. Specificity of Mad-1 protein binding. RNAs encoding theproteins given at the top of each panel were translated and labeled with³⁵ S-methionine in vitro in the presence of either purifiedglutathione-s-transferase (GST) or glutathione-s-transferase fused inframe to baboon Mad-1 cDNA sequence encoding the c-terminal 186 aminoacids of the Mad-I protein (GST-Mad). The proteins bound by GST orGST-Mad were analyzed on SDS polyacrylamide gels. The lanes marked -indicate translation products obtained in the absence of added purifiedprotein. In FIG. 16B the arrows mark the position of either the ABR Maxor Max9 and ALZ Max polypeptide The position of molecular weight markers(in kD) are given at the right of each panel.

EXAMPLE 8 The Mad-1:Max Heterodimer Binds DNA Specifically

The Max homodimer and the Myc:Max heterodimer bind the sequence CACGTG(CM- 1). We wondered if Mad-1 alone or with Max could recognize the samesequence. Using the electrophoretic moblility shift assay purified(EMSA) GST-mad or GST alone were tested in the presence or absence ofMax for binding to the CM- I oligo (FIG. 17A).

In the absence of Max no binding was detected. This rules outnonspecific interaction of the GST portion of the fusion protein withDNA and suggests, as above, that mad homodimers did not form or were notstable. In the presence of Max a new slower migrating protein-DNAcomplex was seen in the presence of GST-Mad. Again, GST protein alonehad no effect. The new complex was caused by binding of the GST-Mad:Maxheterodimer because it was further retained in the gel matrix byantibodies specific for Max or GST. This supershift was reversible whenthe corresponding immunogen was added to the binding reaction andtherefore specific for the given antibody-antigen complex. Mad:Maxbinding of the CM-1 oligo was specific because its activity in the EMSAwas competed by cold CM-1 but not by equivalent amounts of a unrelatedoligo of similar length (data not shown). We also wanted to investigatethe binding of a potential Mad-1:Myc complex binding to the CM-1 oligo.Consistent with the lack of interaction between GST-C92Myc and GST-Mad(FIG. 16C) there was little or no DNA binding from a putativeGST-Mad:GST-C92Myc heterodimer under conditions where the GST-C92Myc:Maxand the GST-Mad:Max heterodimers form and bind DNA (FIG. 17C).

FIGS. 17A-C. Specific DNA binding by the Mad:Max heterodimer. Theability of Mad-1 to bind DNA and interact with Max and Myc was examinedby the EMSA. Purified proteins, GST, GST, GST-Mad, and GST-C29Myc weretested alone or in the presence of Max for binding to the CM-1oligonucleotide. Which protein(s) was present in the binding reaction isindicated at the top of FIGS. 17A-B. In each experiment the specificityof the mobility shift was assayed by including antibodies to eitherMax((xMax), GST((xGST) or Myc((xMyc) were added to the binding reaction.The activity of these antibodies was inhibited by adding the appropriatein-imunogen to the binding reaction (+block). The lanes marked - had noadditional protein present in the binding reaction. The position of eachprotein-DNA complex and the unbound probe is given. The asteriskindicates inclusion of antibody in the complex. A diagram of the GSTproteins used in this experiment are shown.

EXAMPLE 9 Either GST-C92Myc:Max or GST-Mad:Max are Favored Over Max:MaxHomodimers

The data presented in FIGS. 17A-C suggest that either GST-C92Myc:Max orGSTMad:Mad heterodimers bind the CM-1 site more tightly than the Max:Maxhomodimer. However, at the concentrations of protein used in thatexperiment the DNA probe is nearly exhausted and therefore the observedDNA binding was not in the linear range. To answer this questiondirectly we assayed the amount of DNA binding derived from increasingamounts of Max in the presence or absence of either GST-C92Myc orGST-Mad. Under conditions where there was little or no Max homodimer DNAbinding the addition of either GST-Myc or GST-Mad results in significantheterodimer DNA binding (FIG. 17A and 17B). As the amount of Max in thebinding reaction was increased the heterodimeric binding increased untilsaturation was reached. This result suggested that either theGST-C92Myc:Max or the GSTMad:Max heterodimer was favored over the Maxhomodimeric complex. Based on current models it is most likely that theincreased DNA binding of the heterodimeric species reflected increasedlevels of heterodimers rather than heterodimers having increased DNAbinding activity per se.

EXAMPLE 10 DNA Binding of the Mad:Max Heterodimer Is Not Affected byPhosphorylation

The activity of many transcription factors is regulated bypost-translational modification. DNA binding of Mad homodimers but notMyc:Max heterodimers is blocked by CKII phosphorylation. We wished todetermine what effect CKII phosphorylation had on the DNA bindingactivity of the GST-Mad:Max heterodimer. Purified Max was treated withCKII in the presence or absence of ATP and assayed for DNA alone or withGST or GST-Mad. DNA binding activity tested by the EMSA (FIG. 18).

FIGS. 18A-B. Either the Mad:Max or the Myc:Max heterodimer is favoredover the Max:Max homodimer. Increasing amounts of Max were assayed forDNA binding to the CM- I oligonucleotide by the EMSA either alone or inthe presence of 30 ng GSTMad (FIG. 18A) or 25 ng GST-C92Myc (FIG. 18B).When assayed alone max in the binding reactions was increased in roughly2 fold increments from 0.3 ng to 10 ng. The same amounts of Max weretested with the indicated amount of fusion protein. In the lane marked-- there was no protein in the binding reaction. The positions of theunbound probe and the protein:DNA complexes are indicated.

There was no effect of GST or CKII in the absence of ATP on the Maxhomodimer mobility shift. As before, the addition of GST-Mad to Maxresulted in the slower heterodimer mobility shift which was not effectedby CKII in the absence of ATP. The addition of both CKII and ATP to thebinding reaction resulted in a complete loss of Max homodimer bindingbut the binding of the Mad:Max heterodimer was unaffected. As expectedthe loss of Max homodimer binding was not restored by GST alone.Therefore, like the Myc:Max heterodimer, the GST-Mad:Max heterodimer canover ride the effect of CKII phosphorylation on Max.

EXAMPLE 11

Mad Competes for Max Cmplexed to Myc and Vice Versa

Because the GST-Mad fusion protein only encoded the C-terminal 186 aminoacids of the Mad-1 protein and the fact that the GST-Mad:Max DNA complexcomigrated with the GST-Myc:Max DNA complex in the EMSA we have madeconstruct which encodes the full length Mad-1 coding region fused inframe to six histidines. This fusion protein, which we will refer to asMad-1 throughout, was purified from E. coli by nickel chelatechromatography. The full length histidine tagged Mad-1 protein behavedidentically to the GST-Mad fusion protein. It was able to form aheterodimer with Max capable of specific DNA binding (FIG. 19 and datanot shown).

The Mad:Max heterodimer and the Max homodimer DNA complexes migrated asa closely spaced doublet. The Mad:Max complex is the faster migrating ofthe two protein:DNA complexes. As expected, Mad-I could neither bindCM-I in the absence of Max nor in the presence of GST-C92Myc.

Both Myc and Mad-1 can bind Max. It is possible that one of theheterodimers is more stable than the other and would be expected topredominate even in the presence of the other Max binding partner. Inorder to test this we assayed the stability of one heterodimer byallowing it to form in the presence of the other Max binding partner.Both GSTC92Myc and Mad-1 were greater than 50% pure as judged fromCoomassie stained gels (data not shown); however, it was difficult todetermine the amount of active protein in each preparation. We thereforefirst determined the minimal amount of each fusion protein that wouldcomplex with 2 ng of purified Max (data not shown). In order todetermine the stability of the GST-C92Myc:Max heterodimer complex (in aratio of 6 ng:2 ng) it was allowed to form in the presence of increasingamounts of Mad-1. The converse experiment was done to test the stabilityof the Mad:Max (7.5 ng:2 ng) heterodimer by allowing the complexes toform the presence of increasing amounts of GST-C92Myc. The resultingprotein-DNA complexes were resolved on nondenaturing gels (FIG. 19).

FIG. 19. CKII phosphorylation does not affect the DNA binding of theMad:Max heterodimer. Max or Max treated with CKII was tested for DNAbinding to the CM-1 oligonucleotide by the EMSA presence of GST orGST-Mad. The proteins in the binding reactions are given at the top ofthe figure. In the lanes marked CKII-ATP or CKII+ATP Max was treatedwith CKII either in the absence or the presence of ATP, respectively,prior to inclusion in the DNA binding reaction. The positions of thefree probe and the protein DNA complexes are indicated.

FIGS. 20A-C. The Myc:Max and the Mad:Max DNA binding complexes havesimilar affinities for DNA. The DNA binding characteristics of thepurified histidine tagged Mad was assayed by the EMSA (FIG. 20A). In allthree panels the proteins present in the binding reactions is given atthe top of FIG. 20. ("-") indicates the absence of protein in thebinding reacfion. The positions of the protein:DNA complex and theunbound probe is given at the right of each panel. In the experimentshown in FIG. 20B and FIG. 20C a constant amount of eitherGST-C92Myc:Max (6ng:2ng) (FIG. 20B) or Mad:Max (7.5ng:2ng) (FIG. 20C)was challenged with increasing amount of Mad or GST-C92Myc,respectively. The challenging protein was increased in 2 fold incrementsfor both Mad and GST-C92Myc. The titration started at 1.8 ng for Mad andat 1.6 ng for GST-C92Myc and went to 30 ng and 50 respectively.

The amount of Mad:Max heterodimer complex observed was reduced in thepresence of increasing amounts of GST-C92Myc. A concomitant increase inthe amount of GSTC92Myc:Max complex was also observed. The analogousresult was obtained when GST-Myc:Max complexes were tonned in thepresence of increasing amounts of Mad-1, i.e. there was a decrease inthe amount of GST-C92Myc:Max complex and an increase in the amount ofMad:Max complex. In both experiments there was competition even at thelowest amount of added competing protein (1.6ng of Mad-1 and 1.8 ng forGST-C92 Myc). Similar results were obtained if one pair heterodimerswere allowed to form prior to the addition of the other fusion protein.Although, a higher level of the competing protein was required todisassociate the preformed heterodimeric complex. Given that the halflife of the Myc protein in cells is very rapid it is not clear if thisresult is meaningful. In addition, the DNA binding site did notstabilize or destabilize either of the heterodimeric complexes (data notshown). Because the fusion proteins have roughly the same molecularweight the computation was carried out under conditions with each of theproteins present in near equal molar concentrations. Therefore, theresults suggest that Myc and Mad-1 bind Max with similar affinities andthat the levels of each protein were important in determining whichheterodimer predominated.

DISCUSSION

We present in EXAMPLES 6-11, above, the identification andcharacterization of a new Max dimerization partner:Mad. Mad is a newmember of the bHLH-Zip family of transcription factors and itsinteraction with Max appears to be fairly specific. The physicalcharacteristics of Mad and the Mad:Max heterocomplex are very similar tothose of Myc and the Myc:Max complex. A model for these interactions andtheir outcome on DNA binding are given in FIG. 21.

FIG. 21. A scheme for the interactions between Myc, Max and Mad-1. Thein vitro interactions between Myc Max, and Mad- 1 their specific DNAbinding site are shown.

Myc and Mad bind DNA specifically only at high protein concentrationssuggesting that both proteins homodimerize poorly. Both proteins canheterodimerize with Max to form a specific DNA binding complex. Whilethe model shows only binding to a CACGTG site both heterodimers showidentical binding on variants of this site (K. Blackwell and D. Ayer,unpublished observations). Therefore, while there has been no Mycresponsive cellular target yet identified, it is likely that the in vivobinding sites for the Myc:Max heterodimer will be occupied by Mad:Maxheterocomplex as well. Either the Myc:Max or Mad:Max heterocomplex isfavored over the Max homodimer. Finally, in contrast to the DNA bindingof Max homodimers, the DNA binding activity of either Max containingheterocomplex is unaffected by CKII phosphorylation. It will beinteresting to determine if Mad and Myc are similar in othercharacteristics. For example, is Mad a unstable protein and regulatedupon entry to and exit from the cell cycle.

In transient transfection assays, Myc activated transcription of aheterologous reporter gene containing the CACGTG binding motif in itspromoter while Max repressed transcription from the reporter construct(Kretzner et. al, submitted). Because Myc forms heterodimers poorly itwas assumed that interaction with Max was responsible for the observedMyc transcriptional activation. Max repression has been assumed to bethe result of Max homodimers repressing transcription from the reporterconstruct (FIRST AND SECOND SERIES OF EXAMPLES, above). It must now alsobe considered that transciptional repression observed may in certaincases have been due to Max interacting with endogenous Mad. If Myc:Maxis responsible for transcriptional activation and Mad:Max potentiallyacts as a transcriptional repressor, what function do Max homodimersplay in controlling gene expression? Because Max homodimers areunfavored, in the presence of both Myc and Mad, the cellular levels ofMax homodimers will be low. In addition, cells have evolved a mechanismto keep Max homodimers off DNA, namely CKII phosphorylation. Maxisolated from cells is phosphorylated 0 and treatment of purified Maxfrom human or insect cells with aluine phosphatase greatly increases DNAbinding (D. Ayer and E. Blackwood, unpublished observation). These datasuggest that the majority of Max is either off the DNA as homodimers orbound as either a Myc:Max or Mad:Max heterocomplex.

By binding to Max, Mad potentially antagonizes Myc function. In vitrobinding experiments suggest that the relative levels of either Myc orMad will determine which of the heterocomplexes will be most prevalent.It is therefore critical to determine levels of Myc and Max protein invarious cells and times during the cell cycle. If the Mad:Max complex isthe antithesis of the Myc:Max complex one might expect Mad expression tobe highest where Myc activity is low. For example, Mad expression mightbe elevated in resting G₀ cells or cells that are terminallydifferentiated.

Max allows two proteins, Myc and Mad, to bind DNA raising thepossibility that other proteins might also require Max to mediate theirDNA binding and activity. We are cur-rently screening expressionlibraries for other Max binding proteins. Interestingly, using a geneticscreen in yeast, another lab has cloned a CDNA encoding a protein,distinct from Mad, but having properties very similar to those of Mad(T. Zervos, personal communication).

EXPERIMENTAL PROCEDURES

Expression and purification of Max. The cDNA encoding Max was clonedinto the baculovirus transfer vector pJVNhe and recombinant virus wasplaque purified as described. For Max expression and purification 2×10⁸Sf9 cells were infected at an MOI of 5. 48 hours PI cells were washedtwice with PBS and a cytoplasmic extract was prepared by lysing thecells in HMO.1* (50 mM Hepes pH 7.5, 100 mM KCl, 10 mM MgCl₂, 1 mM EDTA,10% glycerol, 1 mM DTT, 0.5% NP-40, 10 mg/ml PMSF and 0.1% aprotinin)and pelleting the cellular debris at 10,000xg for 10 minutes. Maxrepresented about 1% of the total protein present in this cytoplasmicextract. The cytoplasmic extract was applied to a 25 ml BioRex 70(Biorad) column equilibrated in HMO.1 (identical to HMO.1* but with noNP-40). Max was eluted with a linear gradient of KCl in HMO.1. Themajority of the Max eluted in a broad peak between 0.3 and 0.6M KCl. TheMax containing fractions were pooled and diluted with HMO (HMO.1 with noKCl) to a conductivity corresponding to 100 mM KCl and applied to amono-Q HR 5/5 column (Pharmacia) equilibrated in HMO.1. Max elutedbetween 200 and 300 mM KCl. The peak fractions were pooled and stored at-80° C. This Max preparation was at least 95% pure as judged by silverstaining of SDS polyacrylamide gels.

Screening for Max Binding partners: 3 μg purified Max protein waslabeled to high specific activity (10⁷ cpm/μg) by CKII (a kind gift ofD. Litchfield and E. Krebs) and [γ-³² P] ATP (3000 Ci/mMol, Dupont NEN)in HMO.1 for 30 minutes at 30° C. The unincorporated ATP was removed ona Sephadex G-50 column equilibrated with HMO.1. Approximately 10⁶ phageform the 594S gt11 expression library were plated in LE392 on 150 mm×15mm dishes and incubated at 37° C. After the plaques had reached 2-3 mmin size filters (Amersham Hybond C Extra) were overlaid and the proteinswere allowed to transfer overnight at 37° C. The filters were blocked (4washes of 30 minutes each at 4° C.) in 5% nonfat dry milk, 20 mM HepespH 7.5,50 mM KCl, 10 mM MgCl₂, 10 mM β glycerol phosphate, 1 mM DTT and0.1% NP-40. Filters were probed for 4-5 hours with 3×10⁷ cpm Max probe(roughly 5 nM Max) in 1% nonfat dry milk, 20 mM Hepes pH 7.5, 50 mM KCl,10 mM MgCl₂, 10 mM β glycerol phosphate, 10 mM DTT, 0.1% NP-40 and 10%glycerol. The filters were washed in PBS/0.2% Triton X-100 for 10minutes to remove the bulk of the unbound counts. Three additionalwashes (15-30 minutes each) were done with PBS/0.2% Triton X-100+100 mMKCl. The filters were wrapped in saran and exposed for autoradiography.The phage DNA from the positive clones was purified (Qiagen Lambda minikit) and the cDNA insert cloned into the vector pVZ-1.

Screening for the human Mad homolog: A fragment from the 5' end of thebaboon Mad cDNA was isolated and used to probe a human gt10 cDNA libraryfrom embryonic lung fibroblasts (WI-26). Two positive clones wereidentified. The phage DNA was purified and the cDNA insert subclonedinto pVZ-1. All DNA sequencing was done using the Sequenase kit fromUnited States Biochemical.

Production and purification fusion proteins: An oligo(TCAGAATTCTATACAAAAGG) was synthesized that overlapped the 5'end of thebaboon Mad cDNA and use with T3 promoter primer to PCR amplify the MadcDNA. The amplified DNA fragment was subcloned into pGEX-2T. Cellscontaining the pGEX-2TMad plasmid were grown to OD600 of 0.7 and inducedwith 0.2 mM IPTG and the fusion proteins purified as described with thefollowing modification. The fusion protein was eluted HMO.1 containing 5mM glutatione (Sigma). The GST-C92 Myc fusion protein produced in asimilar fashion. To make a cDNA encoding the Mad protein fused to 6histidines full length human Mad-1 coding region was amplified using theT7 promoter primer and a oligo(TCAGTCCATGGCTAGTGGTGGTGGTGGTGGTGGAGACCAAGACACGC) that overlapped the3'end of the Mad coding region but also encoded six in frame histidinesand a stop codon. The amplified product was cloned into pET11D andintroduced in the E. coli strain DE3. The Mad-Ifusion protein waspurified using nickel chelate chromatography under denaturing conditionsaccording to the vendor's instructions (Qiagen) and dialyzed againstthree changes of HMO.1. All proteins were stored at -80° C.

Subcloning of the human Mad cDNA: The 3' untranslated region of the MadcDNA was removed using an internal Eco R1 site and the Eco R1 site inthe polylinker of pVZ-1. Because the Mad cDNA was translated poorly invitro it was necessary to remove the 5' untranslated sequences. Ilis wasdone by PCR using an oligo (GTCAGAATTCACCATGGCGGCGGCGGTT) to the 5'endof the Mad-1 coding region and the T3 promoter primer. The amplifiedproduct was cloned back into pVZ-1 and into the vector pcite-1(Novogen).

In vitro characterization of Mad binding partners: Various cDNAs weretranslated in the presence of 50 ng GST-Mad or 50 ng GST. Followingtranslation, 50 μl of PBS containing an additional 100 mM KCI and 0.5%NP-40 was added along with glutathione-sepharose beads (Pharmacia). Thetranslation products were allowed to bind for 10 minutes and the beadswere washed 5 times with PBS containing 100 mM KCl and 0.5% NP-40. Thebound proteins were eluted with sample buffer and analyzed on SDSpolyacrylamide gels.

Electrophoretic mobility shift assays: The binding reactions contained0.2-0.5 ng of the CM-1 binding site (CCCCCACCACGTGGTGCCTGA) 25 mM HepespH 7.5, 50 mM KCl, 5 mM MgCl₂,0.5 mM EDTA, 5% glycerol, 10 mM DTT, 0.1%NP-40, 0.5 mg/ml BSA, 0.02% Bromophenol blue/xylene cyanol and theindicated amount of protein. In the cases where the amount of protein inthe binding reaction is not given typically 5-10 ng Max or 20-30 ng ofthe various fusion proteins were used. The resulting protein DNAcomplexes were resolved on 5% polyacrylamide gels run with 25 mM HepespH 7.5 in the gel and the running buffer. The gels were run in at 4° C.The non-specific site used in the competitions corresponded to the MREAsite in the Mim-1 promoter (TCGAGTAAGACACCCGTTACTTTACG). CKIIphosphoryation of Max was performed using 3 ng purified CKII in the gelshift buffer given above plus 100 mM ATP.

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While the preferred embodiment of the invention has been illustrated anddescribed, it is to be understood that, within the scope of the appendedclaims, various changes can be made

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES:6                                                   (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:537 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:double                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:cDNA to mRNA                                               (A) DESCRIPTION:Human helix-loop- helix zipper protein (max)                   mRNA, start=position 1                                                       (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:Homo sapiens; Eukaryota; Animalia; Metazoa;                      Chordata; Vertebrata; Mammalia; Theria; Eutheria;                             Primates; Haplorhini; Catarrhini; Hominidae.                                  (vii) IMMEDIATE SOURCE:Human lymphoid B cell Manca cell line,                 cDNA to mRNA                                                                  (ix) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAGTGGCCGCTCCCTGGGCCGTAGGAAATGAGCGATAACGATGACATCGAG GTGGAGAGC60               GACGAAGAGCAACCGAGGTTTCAATCTGCGGCTGACAAACGGGCTCATCATAATGCACTG120               GAACGAAAACGTAGGGACCACATCAAAGACAGCTTTCACAGTTTGCGGGACTCAGTCCCA180               TCACTCCAAGGAGAGAAGGCATCCCGGGCCCAAATCCT AGACAAAGCCACAGAGTATATC240              CAGTATATGCGAAGGAAAAACCACACACACCAGCAAGATATTGACGACCTCAAGCGGCAG300               AATGCTCTTCTGGAGCAGCAAGTCCGTGCACTGGAGAAGGCGAGGTCAAGTGCCCAACTG360               CAGACCAACTACCCCTCCTCAGA CAACAGCCTCTACACCAACGCCAAGGGCAGCACCATC420              TCTGCCTTCGATGGGGGCTCAGACTCCAGCTCAGAGTCTGAGCCTGAAGAGCCCCAAAGC480               AGGAAGAAGCTCCGGATGGAGGCCAGCTAAGCCACTCGGGGCAGGCCAGCAATAAAA537                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:510 base pairs                                                     (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:double                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:cDNA to mRNA                                               (A) DESCRIPTION:Human helix-loop- helix zipper protein                        (max) mRNA, alternative sequence corresponds to                               positions 1- 63 and 91-537 in SEQ. ID. NO. 1.                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:Homo sapiens; Eukaryota; Animalia; Metazoa;                      Chordata; Vertebrata; Mammalia; Theria; Eutheria;                             Primates; Haplorhini; Catarrhini; Hominidae.                                  (vii) IMMEDIATE SOURCE:Human lymphoid B cell Manca cell line,                 cDNA to mRNA                                                                  (ix) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAGTGGCCGCTCCCTGGGCCGTAGGAAATGAGCGATAACGATGACATCGAGGTGGAGAGC60                GACGCTGACAAACGGGCTCATCA TAATGCACTGGAACGAAAACGTAGGGACCACATCAAA120              GACAGCTTTCACAGTTTGCGGGACTCAGTCCCATCACTCCAAGGAGAGAAGGCATCCCGG180               GCCCAAATCCTAGACAAAGCCACAGAGTATATCCAGTATATGCGAAGGAAAAACCACACA240               CACCAGCAA GATATTGACGACCTCAAGCGGCAGAATGCTCTTCTGGAGCAGCAAGTCCGT300              GCACTGGAGAAGGCGAGGTCAAGTGCCCAACTGCAGACCAACTACCCCTCCTCAGACAAC360               AGCCTCTACACCAACGCCAAGGGCAGCACCATCTCTGCCTTCGATGGGGGCTCAGACTCC 420              AGCTCAGAGTCTGAGCCTGAAGAGCCCCAAAGCAGGAAGAAGCTCCGGATGGAGGCCAGC480               TAAGCCACTCGGGGCAGGCCAGCAATAAAA510                                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:160 amino acids                                                    (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:polypeptide                                                (A) DESCRIPTION:Human helix-loop- helix zipper protein; amino                 acid sequence predicted SEQ.ID.NO. 1                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:Homo sapiens; Eukaryota; Animalia; Metazoa;                      Chordata; Vertebrata;Mammalia; Theria; Eutheria;                              Primates; Haplorhini; Catarrhini; Hominidae.                                  (vii) IMMEDIATE SOURCE:Human lymphoid B cell Manca cell line                  (ix) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetSerAspAsn AspAspIleGluValGluSerAspGluGluGlnPro                             151015                                                                        ArgPheGlnSerAlaAlaAspLysArgAlaHisHisAsnAlaLeuGlu                              20 2530                                                                       ArgLysArgArgAspHisIleLysAspSerPheHisSerLeuArgAsp                              354045                                                                        SerValProSerLeuGlnGlyGluLysAlaSerArg AlaGlnIleLeu                             505560                                                                        AspLysAlaThrGluTyrIleGlnTyrMetArgArgLysAsnHisThr                              65707580                                                                      Hi sGlnGlnAspIleAspAspLeuLysArgGlnAsnAlaLeuLeuGlu                             859095                                                                        GlnGlnValArgAlaLeuGluLysAlaArgSerSerAlaGlnLeuGln                               100105110                                                                    ThrAsnTyrProSerSerAspAsnSerLeuTyrThrAsnAlaLysGly                              115120125                                                                     SerThrIleSerAlaPheAspGlyG lySerAspSerSerSerGluSer                             130135140                                                                     GluProGluGluProGlnSerArgLysLysLeuArgMetGluAlaSer                              145150155 160                                                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:151 amino acids                                                    (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:polypeptide                                                (A) DESCRIPTION:Human helix-loop- helix zipper protein; amino                 acid sequence predicted SEQ. ID. NO. 2                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:Homo sapiens; Eukaryota; Animalia; Metazoa;                      Chordata; Vertebrata; Mammalia; Theria; Eutheria;                              Primates; Haplorhini; Catarrhini; Hominidae.                                 (vii) IMMEDIATE SOURCE:Human lymphoid B cell Manca cell line                  (ix) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetSerAspAsnAspAspIleGluValGluSerAspAlaAspLysArg                              151015                                                                        AlaHisHisAsn AlaLeuGluArgLysArgArgAspHisIleLysAsp                             202530                                                                        SerPheHisSerLeuArgAspSerValProSerLeuGlnGlyGluLys                              35 4045                                                                       AlaSerArgAlaGlnIleLeuAspLysAlaThrGluTyrIleGlnTyr                              505560                                                                        MetArgArgLysAsnHisThrHisGlnGlnAspIleAspAspLeu Lys                             65707580                                                                      ArgGlnAsnAlaLeuLeuGluGlnGlnValArgAlaLeuGluLysAla                              859095                                                                        A rgSerSerAlaGlnLeuGlnThrAsnTyrProSerSerAspAsnSer                             100105110                                                                     LeuTyrThrAsnAlaLysGlySerThrIleSerAlaPheAspGlyGly                              115 120125                                                                    SerAspSerSerSerGluSerGluProGluGluProGlnSerArgLys                              130135140                                                                     LysLeuArgMetGluAlaSer                                                         145 150                                                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:1002 base pairs                                                    (B) TYPE:nucleic acid                                                         (C) STRANDEDNESS:single                                                       (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:cDNA                                                       (A) DESCRIPTION:FIGURE 14                                                     (ix) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CGCCAGAGAGGCTCCCTCAGCCCTGCTCCGCGGGGTCCACAGCGGGC TCCACAGCGGGCT60               CCATAGCGGGCTCCACAGCGGTCCGGCGGCGGCAGCGAGCCCGTGGGCAGTGGGGGTTGG120               TCCCGTGGCTCCGGCCCCCGGTGCAGAATGGCGGCGGCGGTTCGGATGAACATCCAGATG180               CTGCTGGAGGCGGCCGACTATCTGGAGCGGCG GGAGAGAGAAGCTGAACATGGTTATGCC240              TCCATGTTACCATACAATAACAAGGACAGAGATGCCTTAAAACGGAGGAACAAATCCAAA300               AAGAATAACAGCAGTAGCAGATCAACTCACAATGAAATGGAGAAGAATAGACGGGCTCAT360               CTTCGCTTGTGCCTGGAG AAGTTGAAGGGGCTGGTGCCACTGGGACCCGAATCAAGTCGA420              CACACTACGTTGAGTTTATTAACAAAAGCCAAATTGCACATAAAGAAACTTGAAGATTGT480               GACAGAAAAGCCGTTCACCAAATCGACCAGCTTCAGCGAGAGCAGCGACACCTGAAGAGG540               CAG CTGGAGAAGCTGGGCATTGAGAGGATCCGGATGGACAGCATCGGCTCCACCGTCTCC600              TCGGAGCGCTCCGACTCCGACAGGGAAGAAATCGACGTTGACGTGGAGAGCACGGACTAT660               CTCACAGGTGATCTGGACTGGAGCAGCAGCAGTGTGAGCGACTCTGACGAGCGG GGCAGC720              ATGCAGAGCCTCGGCAGTGATGAGGGCTATTCCAGCACCAGCATCAAGAGAATAAAGCTG780               CAGGACAGTCACAAGGCGTGTCTTGGTCTCTAAGAGAGTGGGCACTGCGGCTGTCTCCTT840               GAAGGTTCTCCCTGTTGGTTCTGATTAGGTAACGTATTGG ACCTGCCCACAACTCCCTTG900              CACGTAAACTTCAGTGTCCCACCTTGACCAAAATCAGCTTTGTAACTGTTTTCAAGGAGG960               TGCTTAGGATTGTGGGTTTCTGATTGCATCACTAGCTTCTCC1002                                (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:221 amino acids                                                     (B) TYPE:amino acid                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:polypeptide                                                (A) DESCRIPTION:Amino acid sequence from Seq ID No.1 from                     base 148 to 810; FIGURE 14                                                    (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (ix) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetAlaAlaAlaValArgMetAsnIleGlnMetLeuLeuGluAla                                 1 51015                                                                       AlaAspTyrLeuGluArgArgGluArgGluAlaGluHisGlyTyr                                 202530                                                                        AlaSerMetLeuPr oTyrAsnAsnLysAspArgAspAlaLeuLys                                354045                                                                        ArgArgAsnLysSerLysLysAsnAsnSerSerSerArgSerThr                                 50 5560                                                                       HisAsnGluMetGluLysAsnArgArgAlaHisLeuArgLeuCys                                 657075                                                                        LeuGluLysLeuLysGlyLeuValProLeuGlyProG luSerSer                                808590                                                                        ArgHisThrThrLeuSerLeuLeuThrLysAlaLysLeuHisIle                                 95100105                                                                      Lys LysLeuGluAspCysAspArgLysAlaValHisGlnIleAsp                                110115120                                                                     GlnLeuGlnArgGluGlnArgHisLeuLysArgGlnLeuGluLys                                 125 130135                                                                    LeuGlyIleGluArgIleArgMetAspSerIleGlySerThrVal                                 140145150                                                                     SerSerGluArgSerAspSerAspAr gGluGluIleAspValAsp                                155160165                                                                     ValGluSerThrAspTyrLeuThrGlyAspLeuAspTrpSerSer                                 170175 180                                                                    SerSerValSerAspSerAspGluArgGlySerMetGlnSerLeu                                 185190195                                                                     GlySerAspGluGlyTyrSerSerThrSerIleLysArgIleLys                                  200205210                                                                    LeuGlnAspSerHisLysAlaCysLeuGlyLeu                                             215220                                                                    

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. An isolated nucleic acidmolecule capable of hybridizing under stringent conditions to thenucleotide sequence residing between positions 1 and 1002 of the madcDNA shown in FIG. 14 (Seq. I.D. No. 5).
 2. The isolated nucleic acidmolecule of claim 1, encoding a Mad polypeptide capable of associatingwith a Max polypeptide.
 3. The isolated nucleic acid molecule of claim1, encoding a polypeptide capable of binding to an antibody that bindsto the Mad polypeptide shown in FIG. 14 (Seq. I.D. No: 6).
 4. Arecombinant expression vector comprising the isolated nucleic acidmolecule of claim 1 operably linked to suitable control sequences. 5.Cells transfected or transduced with the recombinant expression vectorof claim
 4. 6. A method of producing a Mad polypeptide comprisingculturing cells of claim 5 to produce a Mad polypeptide that associateswith a Max polypeptide.
 7. An isolated DNA molecule capable ofhybridizing under stringent conditions to the nucleotide sequenceresiding between positions 355 and 399 of the helix I region of the MadcDNA shown in FIG. 14 (Seq. I.D. No.: 5).
 8. An isolated DNA moleculecapable of hybridizing under stringent conditions to the nucleotidesequence residing between positions 418 and 471 of the helix II regionof the mad cDNA shown in FIG. 14 (Seq. I.D. No: 5).
 9. An isolated DNAmolecule comprising the basic region sequence residing between positions319 and 354 of the mad cDNA shown in FIG. 14 (Seq. I.D. No.: 5).
 10. Anisolated DNA molecule encoding a polypeptide capable of associating witha Max polypeptide and inhibiting binding of Max to a nucleotide sequencecomprising CACGTG, the isolated DNA molecule capable of hybridizing tothe nucleotide sequence residing between positions 354 and 472 shown inFIG. 14 but not to the basic region residing between positions 319 and354 shown in FIG. 14 (Seq. I.D. No.: 5).